Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures.

Madin-Darby canine kidney (MDCK) cells form polarized, multicellular epithelial structures in vitro. Low-level expression of pp60v-src in MDCK cells elicits plasticity in these multicellular structures. Plasticity was revealed by the displacement of cells from mechanically stressed regions of the epithelial monolayers; however, the two-dimensional relationship between the cells in the remainder of the monolayer was maintained. Electron microscopy of multicellular structures revealed abnormal separation of the lateral membranes of adjacent cells and selective uncoupling of the junctional complex; the zonula adherens was disrupted, but the zonula occludens and desmosomes were retained. Significantly, this result was not accompanied by transformation of the cells, as judged by the absence of anchorage-independent growth potential. These results demonstrate a nonmitogenic biological activity of pp60v-src which is experimentally dissociable from transformation. This morphoregulatory action on higher-order epithelial structures may reflect a function of related cellular tyrosine kinases.

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Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin.

The Journal of Cell Biology ◽

10.1083/jcb.101.4.1307 ◽

1985 ◽

Vol 101 (4) ◽

pp. 1307-1315 ◽

Cited By ~ 168

Author(s):

J Behrens ◽

W Birchmeier ◽

S L Goodman ◽

B A Imhof

Keyword(s):

Monoclonal Antibody ◽

Epithelial Cells ◽

Mdck Cells ◽

Apical Cell ◽

Junctional Complex ◽

Madin Darby Canine Kidney ◽

Surface Antibody ◽

Darby Canine Kidney ◽

Canine Kidney

It has previously been shown that the monoclonal antibody anti-Arc-1 dissociates Madin-Darby canine kidney (MDCK) epithelial cells and changes their morphology in vitro (Imhof, B.A., H.P. Vollmers, S.L. Goodman, and W. Birchmeier, 1983, Cell, 35:667-675). In this article we demonstrate that the anti-Arc-1 antibody recognizes an uvomorulin-like molecule on MDCK cells, i.e., it immunoprecipitates an 84-kD protein fragment from a tryptic digest of cell surfaces in the presence of Ca2+ (as does anti-uvomorulin antiserum). Furthermore, anti-uvomorulin antiserum prevents the binding of anti-Arc-1 to MDCK cells. The distribution of the Arc-1 antigen is also quite similar to that of uvomorulin: it is enriched at the cell-cell contacts both of MDCK cells and of cells in various canine tissues. In the intestinal epithelium the antigen could be further localized in the region of the junctional complex. To study the mechanism of action of the dissociating antibody, MDCK cells grown on Nuclepore filters in Boyden chambers were exposed to anti-Arc-1 from either the upper or lower compartment. It could be shown that the antibody interfered with cell adhesion only from the basolateral but not from the apical cell surface. Antibody action was inhibited in the presence of colchicine but not cytochalasin B. Furthermore, cell dissociation was prevented when the cellular cAMP level was raised. These findings indicate that the anti-Arc-1 antibody acts on a target below the tight junctions (possibly on the antigen located in the junctional complex), and they confirm that cytoskeleton and metabolic factors are actively involved in the maintenance of junctional integrity.

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Transepithelial Resistance and Inulin Permeability as Endpoints in In Vitro Nephrotoxicity Testing

Alternatives to Laboratory Animals ◽

10.1177/026119290203002s08 ◽

2002 ◽

Vol 30 (2_suppl) ◽

pp. 53-59 ◽

Cited By ~ 13

Author(s):

Tracey Duff ◽

Simon Carter ◽

Gemma Feldman ◽

Gordon McEwan ◽

Walter Pfaller ◽

...

Keyword(s):

Proximal Tubule ◽

Electrical Resistance ◽

Mdck Cells ◽

Ussing Chamber ◽

Fluorescein Isothiocyanate ◽

Transepithelial Electrical Resistance ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney

Transepithelial electrical resistance (RT) and the flux of fluorescein isothiocyanate (FITC) across Madin Darby canine kidney (MDCK) strain 1 cells and porcine epithelial kidney (LLC-PK1) monolayers were compared between three laboratories for a range of nephrotoxins. The precision of the REMS AutoSampler was similar to that of the Ussing chamber and the ENDOHM® technique, but superior to using chopstick electrodes, for measurements of resistance. The nephrotoxins used were selective for the proximal tubule, and in all cases, LLC-PK1 cells were more sensitive than MDCK cells. In most cases, change in RT was a more sensitive indicator of damage than alterations in FITC flux. The REMS system provides high intra-plate precision for RT measurements and is a higher throughput system, which is applicable to screening for nephrotoxicity in vitro.

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STAT1 Is Required for Redifferentiation during Madin-Darby Canine Kidney Tubulogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e10-02-0112 ◽

2010 ◽

Vol 21 (22) ◽

pp. 3926-3933 ◽

Cited By ~ 9

Author(s):

Minji Kim ◽

Lucy Erin O'Brien ◽

Sang-Ho Kwon ◽

Keith E. Mostov

Keyword(s):

Epithelial Mesenchymal Transition ◽

Mdck Cells ◽

Madin Darby Canine Kidney ◽

Tubule Formation ◽

Mesenchymal Transition ◽

Phase Loss ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Constitutively Active

Tubule formation in vitro using Madin-Darby canine kidney (MDCK) epithelial cells consists mainly of two processes. First, the cells undergo a partial epithelial–mesenchymal transition (pEMT), losing polarity and migrating. Second, the cells redifferentiate, forming cords and then tubules with continuous lumens. We have shown previously that extracellular signal-regulated kinase activation is required for pEMT. However, the mechanism of how the pEMT phase is turned off and the redifferentiation phase is initiated is largely unknown. To address the central question of the sequential control of these two phases, we used MDCK cells grown as cysts and treated with hepatocyte growth factor to model tubulogenesis. We show that signal transducer and activator of transcription (STAT)1 controls the sequential progression from the pEMT phase to the redifferentiation phase. Loss of STAT1 prevents redifferentiation. Constitutively active STAT1 allows redifferentiation to occur even when cells are otherwise prevented from progressing beyond the pEMT phase by exogenous activation of Raf. Moreover, tyrosine phosphorylation defective STAT1 partially restored cord formation in such cells, suggesting that STAT1 functions in part as nonnuclear protein mediating signal transduction in this process. Constitutively active or inactive forms of STAT1 did not promote lumen maturation, suggesting this requires a distinct signal.

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An In Vitro Model of the Blood-Brain Barrier: The Response of Madin-Darby Canine Kidney Cells to Triethyl Tin

Alternatives to Laboratory Animals ◽

10.1177/026119299602400308 ◽

1996 ◽

Vol 24 (3) ◽

pp. 349-357

Author(s):

Bellina Veronesi ◽

Kent Carlsón ◽

Marion Ehrich

Keyword(s):

Cell Line ◽

Blood Brain Barrier ◽

Electrical Resistance ◽

Mdck Cells ◽

Brain Barrier ◽

Madin Darby Canine Kidney ◽

Blood Brain ◽

Darby Canine Kidney ◽

Canine Kidney

The development of a cell culture model which simulates the properties of the blood–brain barrier (BBB) is necessary for the detection of neurotoxic chemicals that can disrupt the barrier, and to provide a more “risk relevant” in vitro screening battery. The present study evaluates the Madin-Darby canine kidney (MDCK) epithelial cell line for this purpose. Changes in electrical resistance and enzyme activities were correlated in confluent MDCK cells exposed to the neurotoxic metal, triethyl tin (TET). Concentrations of TET (0.001–10μM) were established that produced depression in electrical resistance of the MDCK cells after exposure for 8 hours or caused fluorescein leakage after exposure for 72 hours. Confluent cultures of MDCK cells were then exposed to these concentrations of TET and assayed after exposure for 24 hours and 72 hours for changes in those enzymes common to both epithelial and cerebral endothelial cells. The results indicated that increased alkaline phosphatase (APP), γ-glutamyl transpeptidase (GGTP) and superoxide dismutase (SOD) characterised the loss of electrical resistance and permeability disruption in TET-exposed MDCK confluent cultures. Relative increases in APP and decreases in GGTP activities preceded cytotoxicity, which was associated with a high SOD activity. Such enzyme changes may be predictive endpoints of barrier cell disruption by neurotoxic metals in this cell line and support the additional evaluation of the MDCK cell line as an in vitro “screen” for chemicals that disrupt the BBB.

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IN VITRO THROMBIN DOSE RESPONSE ON MADIN DARBY CANINE KIDNEY CELL MONOLAYER

NANO ◽

10.1142/s1793292011002718 ◽

2011 ◽

Vol 06 (04) ◽

pp. 333-336

Author(s):

RASHID AMIN ◽

IMRAN SHAKIR ◽

ISHRAT SULTANA ◽

SUNG HA PARK ◽

RAFAQAT HUSSAIN

Keyword(s):

Dose Response ◽

Mdck Cells ◽

Cell Monolayer ◽

Madin Darby Canine Kidney ◽

Cytoskeletal Organization ◽

Inflammatory Conditions ◽

Vitro Model ◽

Darby Canine Kidney ◽

Canine Kidney

Epithelial cells are known to play an important role in sustaining the airway barrier that may be impaired in certain inflammatory conditions. Recently, the use of thrombin has been reported to open the airway of patients with asthma as well as enhance the permeability of endothelial cell monolayers. We designed an in vitro model of Madin Darby Canine Kidney (MDCK) cells and the physiological functions of this model were evaluated by measuring the transepithelial resistance (TER). The epithelial cytoskeletal organization was observed by staining with Bisbenzimide and Rodamine-Phalloidin (BBZ-Phalloidin) and confirmed by fluorescence microscopy. Measurements of the TER generated values up to 2020 Ω/cm2. A dose response of thrombin was observed, showing the permeability changes in the MDCK monolayer and subsequent recovery. A relationship between TER values and cytoskeletal organization was also observed and discussed.

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Effects of Surfactant Retreatment in Vitro: A Method to Evaluate Changes in Cell Junctions and in Cell Viability

Alternatives to Laboratory Animals ◽

10.1177/026119299602400512 ◽

1996 ◽

Vol 24 (5) ◽

pp. 859-865

Author(s):

Richard Clothier ◽

Rebekah Sansom

Keyword(s):

Cell Viability ◽

Mdck Cells ◽

Control Level ◽

Cell Junctions ◽

Alamar Blue ◽

Single Treatment ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney

Madin-Darby canine kidney (MDCK) cells form cell junctions in vitro, which can be damaged and restored following exposure to a mild cytotoxicant. Both tight junctional integrity and cell viability can be measured by using the fluorescein leakage assay and the Alamar Blue™ assay, respectively, without terminating the cell cultures. By combining these two assay endpoints, it is possible to examine the effects of a repeated cocamidopropylbetaine treatment on a confluent layer of MDCK cells. While the tight junctions could be restored after a single treatment with 2.5mg/ml or 10mg/ml cocamidopropylbetaine, after a second insult, recovery was noted for those treated with 2.5mg/ml, but not for those exposed to 10mg/ml. Following the second insult, the cells did not regain the control level of viability.

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Microlith Formation In Vitro by Madin Darby Canine Kidney (MDCK) Cells

International Journal of Urology ◽

10.1111/j.1442-2042.1996.tb00624.x ◽

1996 ◽

Vol 3 (1) ◽

pp. 23-26 ◽

Cited By ~ 7

Author(s):

Shinji Kageyama ◽

Yoshihisa Ohtawara ◽

Kimio Fujita ◽

Tetsuya Watanabe ◽

Tomomi Ushiyama ◽

...

Keyword(s):

Mdck Cells ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney

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Effect of Riluzole on Ca2+ Movement and Cytotoxicity in Madin-Darby Canine Kidney Cells

Human & Experimental Toxicology ◽

10.1191/0960327106het641oa ◽

2006 ◽

Vol 25 (8) ◽

pp. 461-469 ◽

Cited By ~ 6

Author(s):

W-C Chen ◽

H-H Cheng ◽

C-J Huang ◽

C-T Chou ◽

S-I Liu ◽

...

Keyword(s):

Mdck Cells ◽

Dependent Manner ◽

Madin Darby Canine Kidney ◽

Concentration Dependent Manner ◽

Intracellular Ca ◽

Pre Treatment ◽

Lateral Sclerosis ◽

Darby Canine Kidney ◽

Canine Kidney

Riluzole is a drug used in the treatment of amyotrophic lateral sclerosis; however, its in vitro action is unclear. In this study, the effect of riluzole on intracellular Ca2- concentration ([Ca2-]i) in Madin-Darby canine kidney (MDCK) cells was investigated using the Ca2--sensitive fluorescent dye, fura-2. Riluzole (100 -500 mM) caused a rapid and sustained increase of [Ca2-]i in a concentration-dependent manner (EC50 = 150 mM). Some 40 and 50% of this [Ca2-]i increase was prevented by the removal of extracellular Ca2- and the addition of La3-, respectively, but was unchanged by dihydropyridines, verapamil and diltiazem. In Ca2--free medium, thapsigargin -an inhibitor of the endoplasmic reticulum (ER) Ca2--ATPase -caused a monophasic [Ca2-]i increase, after which the increasing effect of riluzole on [Ca2-]iwas attenuated by 70%; in addition, pre-treatment with riluzole abolished thapsigargin-induced [Ca2-]i increases. U73122, an inhibitor of phospholipase C (PLC), abolished ATP (but not riluzole)-induced [Ca2-]i increases. At concentrations of 250 and 500 mM, riluzole killed 40 and 95% cells, respectively. The cytotoxic effect of riluzole (250 mM) was unaltered by pre-chelating cytosolic Ca2- with BAPTA. Collectively, in MDCK cells, riluzole rapidly increased [Ca2-]i by stimulating extra-cellular Ca2- influx via an La3--sensitive pathway and intracellular Ca2- release from the ER via, as yet, unidentified mechanisms. Furthermore, riluzole caused Ca2--unrelated cytotoxicity in a concentration-depen-dent manner.

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Repolarization of Na+-K+ pumps during establishment of epithelial monolayers

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.5.c896 ◽

1989 ◽

Vol 257 (5) ◽

pp. C896-C905 ◽

Cited By ~ 45

Author(s):

R. G. Contreras ◽

G. Avila ◽

C. Gutierrez ◽

J. J. Bolivar ◽

L. Gonzalez-Mariscal ◽

...

Keyword(s):

Mdck Cells ◽

Surface Membrane ◽

Asymmetric Distribution ◽

Ion Pumps ◽

Madin Darby Canine Kidney ◽

Epithelial Monolayers ◽

Protein Factor ◽

Basolateral Side ◽

Darby Canine Kidney ◽

Canine Kidney

Madin-Darby canine kidney (MDCK) cells plated at confluence and incubated for 20 h in low (5 microM) Ca2+ have no tight junctions (TJs), and their Na+-K+-ATPase is randomly distributed over the surface. On transfer to normal Ca2+ levels (1.8 mM) ("Ca2+ switch"), TJs and transepithelial resistance develop quickly, trapping a considerable fraction (35%) of the surface Na+-K+-ATPase on the apical (incorrect) side. This misplaced enzyme is subsequently removed from this region or inactivated, demonstrating that polarization proceeds despite TJs. Simultaneously, the amount of Na+-K+-ATPase on the basolateral side increases in a higher proportion (125%), than could be accounted for by relocation of the misplaced apical enzyme. This incorporation is prevented by cycloheximide, ammonium chloride, primaquine, or chloroquine, suggesting that Na+-K+-ATPase originates in an intracellular pool and that its surface insertion requires synthesis of new enzyme or of a protein factor, since it is carried to the surface membrane through a mechanism of exocytosis. In summary, asymmetric distribution of ion pumps depends 1) on polarized insertion of Na+-K+-ATPase as well as 2) on removal or inactivation of misplaced enzyme.

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