A cell line derived from normal dog kidney (MDCK) exhibiting qualities of papillary adenocarcinoma and of renal tubular epithelium

Leighton, Joseph; Estes, Larry W.; Mansukhani, Sunder; Brada, Z

doi:10.1002/1097-0142(197011)26:5<1022::aid-cncr2820260509>3.0.co;2-m

Cited by 166 publications

(82 citation statements)

References 8 publications

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“…As shown in Table 1, the Ras trans- fectant shows a statistically greater number of domes, which are 50% smaller. The relationship of domes to fluid transport, and barrier function, properties described in Table 3, has been discussed previously (Leighton et al, 1970;Lever, 1979;Tanner et al, 1983).Also evidenced in the Ras transfectant were occasional multilayered foci of cells (Figure 2, right, white arrow). In thick section microscopy, this sporadic tendency by the Ras transfectant to multilayer is even better illustrated, and contrasts with the monolayer phenotype of LLC-PK 1 and pBabe (Figure 3).…”

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confidence: 60%

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RasMutation Impairs Epithelial Barrier Function to a Wide Range of Nonelectrolytes

Mullin

Leatherman

Valenzano

et al. 2005

MBoC

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Although ras mutations have been shown to affect epithelial architecture and polarity, their role in altering tight junctions remains unclear. Transfection of a valine-12 mutated ras construct into LLC-PK 1 renal epithelia produces leakiness of tight junctions to certain types of solutes. Transepithelial permeability of D-mannitol increases sixfold but transepithelial electrical resistance increases >40%. This indicates decreased paracellular permeability to NaCl but increased permeability to nonelectrolytes. Permeability increases to D-mannitol (M r 182), polyethylene glycol (M r 4000), and 10,000-M r methylated dextran but not to 2,000,000-M r methylated dextran. This implies a "ceiling" on the size of solutes that can cross a ras-mutated epithelial barrier and therefore that the increased permeability is not due to loss of cells or junctions. Although the abundance of claudin-2 declined to undetectable levels in the ras-overexpressing cells compared with vector controls, levels of occludin and claudins 1, 4, and 7 increased. The abundance of claudins-3 and -5 remained unchanged. An increase in extracellular signal-regulated kinase-2 phosphorylation suggests that the downstream effects on the tight junction may be due to changes in the mitogen-activated protein kinase signaling pathway. These selective changes in permeability may influence tumorigenesis by the types of solutes now able to cross the epithelial barrier. INTRODUCTIONMutations in the three closely related ras genes, H-ras, K-ras, and N-ras, are among the most common mutations found in human cancer, reaching 50% in some types of tumors, such as colorectal carcinoma (Forrester et al., 1987;Andreyev et al., 1997Andreyev et al., , 1998Andreyev et al., , 2001). Vogelstein and colleagues have shown that mutations in K-ras occur early in the development of colon carcinomas (Vogelstein et al., 1988). This dominantly acting mutation in ras results in its constitutive activation and the subsequent triggering of its signaling pathway by influencing GTPase activity. It is clear that mutations in ras contribute both to the recurrence of the disease and to decreased survival (Andreyev et al., 1998). It is less clear, however, what the consequences of ras mutations are during the early stages of tumorigenesis. In this regard, one important issue is the effect of Ras activation on epithelial barrier function, perhaps the most basic of all epithelial functions, and a function shared by all epithelial tissues. Ras can exert fundamental effects on epithelial barrier function through the extracellular signal-regulated kinase-mitogen-activated protein (ERK-MAP) kinase pathway or through direct binding to the tight junction-associated protein, AF-6, which binds to ZO-1, which in turn links the tight junction to the actin cytoskeleton. Activated Ras is known to inhibit interaction between the tight junction-associated proteins, AF-6 and ZO-1, with resultant perturbation of intercellular junctions (Yamamoto et al., 1997). Modulation of tight junctions by G proteins in ge...

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confidence: 60%

Section: Resultsmentioning

confidence: 99%

RasMutation Impairs Epithelial Barrier Function to a Wide Range of Nonelectrolytes

Mullin

Leatherman

Valenzano

et al. 2005

MBoC

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“…Madin-Darby canine kidney (MDCK) cells are a cell culture model for epithelial polarization (3,4). When seeded at low density, MDCK cells undergo a morphological change from a fibroblast-like morphology to a well-polarized epithelium (5) and have recently also been adapted to undergo an epithelial-mesenchymal transition (EMT) (6).…”

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Membrane lipidome of an epithelial cell line

Sampaio

Gerl

Klose

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

453

406

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Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required to generate an apical membrane domain that serves as a protective barrier for the epithelial sheet.epithelial polarity | shotgun lipidomics | high resolution mass spectrometry | Forssman glycosphingolipid | epithelial-mesenchymal transition G ain and loss of epithelial polarity are fundamental processes in tissue differentiation (1, 2). These processes have been extensively studied not only in the context of embryogenesis but in tumor progression and metastasis (1). Although cell polarization necessarily implies major changes in the structure and properties of the cell membranes, the exact changes in the molecular composition of their membrane lipidome remain unknown.Madin-Darby canine kidney (MDCK) cells are a cell culture model for epithelial polarization (3,4). When seeded at low density, MDCK cells undergo a morphological change from a fibroblast-like morphology to a well-polarized epithelium (5) and have recently also been adapted to undergo an epithelial-mesenchymal transition (EMT) (6). Epithelial cells have evolved characteristic apical and basolateral membrane domains, separated by tight junctions, with specific protein and lipid composition (7,8). The unusual robustness of the apical membrane is largely attributable to its special lipid composition (9-11).In this work, we applied shotgun MS for comprehensive and quantitative characterization of mammalian cellular membranes (12). By combining high mass resolution MS, an improved extraction procedure that increases the recovery of polar lipids (13), and the use of internal standard mixtures for absolute quantification, we expanded the lipid repertoire covered by shotgun lipidomics to glycosphingolipids (GSPs). Importantly, the analysis of the GSPs did not require the hydrolysis of glycerophospholipids (GPs) (14), and this simplification allowed us to determine the membrane lipidome with unprecedented comprehensiveness (more than 300 lipid species from 14 different lipid classes) from a single sample of 10 5 cells.We further elucidated how the lipidome is remodeled during the course of MDCK cell epithelial polarization. We found that when these c...

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“…Madin-Darby canine kidney (MDCK) cells represent one of the best studied polarized epithelial cell lines and are derived from canine renal tubular epithelium (8). When grown on a permeable filter, MDCK cells form a well polarized epithelial monolayer, exhibiting apical and basolateral plasma membrane domains with unique compositions (9,10), and well defined cell-cell junctional complexes containing tight junctions (11).…”

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The Exocyst Affects Protein Synthesis by Acting on the Translocation Machinery of the Endoplasmic Reticulum

Lipschutz

Lingappa

Mostov

2003

Journal of Biological Chemistry

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We previously showed that the exocyst complex specifically affected the synthesis and delivery of secretory and basolateral plasma membrane proteins. Significantly, the entire spectrum of secreted proteins was increased when the hSec10 (human Sec10) component of the exocyst complex was overexpressed, suggestive of post-transcriptional regulation (Lipschutz, J. H., Guo, W., O'Brien, L. E., Nguyen, Y. H., Novick, P., and Mostov, K. E. (2000) Mol. Biol. Cell 11, 4259 -4275). Here, using an exogenously transfected basolateral protein, the polymeric immunoglobulin receptor (pIgR), and a secretory protein, gp80, we show that pIgR and gp80 protein synthesis and delivery are increased in cells overexpressing Sec10 despite the fact that mRNA levels are unchanged, which is highly indicative of post-transcriptional regulation. To test specificity, we also examined the synthesis and delivery of an exogenous apical protein, CNT1 (concentrative nucleoside transporter 1), and found no increase in CNT1 protein synthesis, delivery, or mRNA levels in cells overexpressing Sec10. Sec10-GFP-overexpressing cell lines were created, and staining was seen in the endoplasmic reticulum. It was demonstrated previously in yeast that high levels of expression of SEB1, the Sec61␤ homologue, suppressed sec15-1, an exocyst mutant (Toikkanen, J., Gatti, E., Takei, K., Saloheimo, M., Olkkonen, V. M., Soderlund, H., De Camilli, P., and Keranen, S. (1996) Yeast 12, 425-438). Sec61␤ is a member of the Sec61 heterotrimer, which is the main component of the endoplasmic reticulum translocon. By coimmunoprecipitation we show that Sec10, which forms an exocyst subcomplex with Sec15, specifically associates with the Sec61␤ component of the translocon and that Sec10 overexpression increases the association of other exocyst complex members with Sec61␤. Proteosome inhibition does not appear to be the mechanism by which increased protein synthesis occurs in the face of equivalent amounts of mRNA. Although the exact mechanism remains to be elucidated, the exocyst/Sec61␤ interaction represents an important link between the cellular membrane trafficking and protein synthetic machinery.

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A cell line derived from normal dog kidney (MDCK) exhibiting qualities of papillary adenocarcinoma and of renal tubular epithelium

Cited by 166 publications

References 8 publications

RasMutation Impairs Epithelial Barrier Function to a Wide Range of Nonelectrolytes

RasMutation Impairs Epithelial Barrier Function to a Wide Range of Nonelectrolytes

Membrane lipidome of an epithelial cell line

The Exocyst Affects Protein Synthesis by Acting on the Translocation Machinery of the Endoplasmic Reticulum

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