C-Met signalling in an HGF/SF-insensitive variant MDCK cell line with constitutive motile/invasive behaviour

1996 ◽  
Vol 109 (9) ◽  
pp. 2371-2381
Author(s):  
C.P. Webb ◽  
K. Lane ◽  
A.P. Dawson ◽  
G.F. Vande Woude ◽  
R.M. Warn
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Mdck Cell ◽  
Mdck Cells ◽  
Cell Surface Expression ◽  
Cell Phenotype ◽  
Mdck Cell Line

The Met protein is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional growth factor with mitogenic, motogenic and morphogenic properties. A morphologically altered variant of the MDCK cell line, MDCK-1, spontaneously exhibits a number of features associated with a partial HGF/SF-Met induced phenotype (less adhesive colonies in culture, enhanced invasion and motility, nascent tubule formation), but paradoxically does not respond to HGF/SF treatment. Although the overall cell surface expression and distribution of Met were found to be similar in parental MDCK cells and the MDCK-1 cell line, p145met autophosphorylation (+/ HGF/SF) was significantly reduced in MDCK-1 cells in vitro and in vivo when compared with parental MDCK cells. In contrast, EGF induced cell proliferation and EGF receptor autophosphorylation to similar levels in both cell lines. The basal levels of protein tyrosine phosphorylation were higher in MDCK-1 cells when compared with parental MDCK cells, including that of two prominent proteins with molecular masses of approximately 185 kDa and 220 kDa. Moreover, both p185 and p220 are present and tyrosine phosphorylated in Met immunoprecipitates from MDCK-1 cells (+/-HGF/SF), but not parental MDCK cells. In addition, Met immunocomplexes from MDCK-1 cells exhibited an approximately 3-fold increased tyrosine kinase activity in vitro when compared with MDCK cells, correlating with the higher basal levels of total phosphotyrosine. Treatment of MDCK-1 cells with the tyrosine kinase inhibitor herbimycin A reverted the cell phenotype to a more MDCK-like morphology in culture, with a concomitant reduction in the tyrosine phosphorylation predominantly of p220. Taken together these data suggest that aberrations in Met activity and associated signalling render MDCK-1 cells insensitive to HGF/SF, and may also mediate alterations in MDCK-1 cell behaviour.

Assessment of aspects of the toxicity of Clostridium perfringens E-toxin using the MDCK cell line

1996 ◽  
Vol 15 (11) ◽  
pp. 904-908 ◽  
Author(s):  
Christopher D Lindsay
Keyword(s):  
Cell Line ◽  
Clostridium Perfringens ◽  
Mechanism Of Action ◽  
Sodium Azide ◽  
Mdck Cell ◽  
Mdck Cells ◽  
Neutral Red ◽  
Temperature Sensitive ◽  
Mdck Cell Line ◽  
Neutral Red Assay

1 The epithelial Madin Darby Canine Kidney (MDCK) cell line was used to study the toxicity of ∈-toxin from Clostridium perfringens. The epithelial MDCK cell line is known to be sensitive to ∈-toxin of Clostridium perfringens and to investigate its mechanism of action, the neutral red assay has been used to determine the viability of cultures of this cell line. 2 Comparison of the LC 50s obtained at 34°C and 0°C showed that the lethality of ∈-toxin was reduced by 18- fold at the lower temperature. The effect of tempera ture on ∈-toxin lethality is unlikely to be due to reductions in membrane fluidity for the addition of Ca2+ or Mg2+ (2 mM) to buffer containing toxin was without effect. Varying the pH of the toxin-containing buffer from 6.9 to 8.7 did not increase the lethality of the toxin, though the most acidic pH used (5.8) was found to potentiate its action on MDCK cells. 3 The effect of inhibiting endocytosis on the lethality ofe toxin was also investigated by incubating cultures of MDCK cells with and without sodium azide over a range of concentrations of toxin. The co-administra tion of sodium azide did not reduce the toxicity of ∈ toxin, suggesting that energy-dependent uptake pro cesses such as endocytosis were unlikely to be involved in its mechanism of action. The results are, however, consistent with known receptor-based me chanisms of uptake and with other mechanisms of internalisation across the plasma membrane. ∈-toxin thus interacts with cell surfaces by a temperature sensitive mechanism potentiated by low pH.

scholarly journals Characterization of Insulin-Like Growth Factor I Receptors on Madin-Darby Canine Kidney (MDCK) Cell Line

1987 ◽  
Vol 34 (3) ◽  
pp. 339-346 ◽  
Author(s):  
IZUMI SUKEGAWA ◽  
NAOMI HIZUKA ◽  
KAZUE TAKANO ◽  
KUMIKO ASAKAWA ◽  
KAZUO SHIZUME
Keyword(s):  
Growth Factor ◽  
Cell Line ◽  
Mdck Cell ◽  
Insulin Like Growth Factor ◽  
Madin Darby Canine Kidney ◽  
Mdck Cell Line ◽  
Factor I ◽  
Darby Canine Kidney ◽  
Canine Kidney

scholarly journals Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An in Vitro Model for the Outer Blood–Retinal Barrier

2012 ◽  
Vol 9 (11) ◽  
pp. 3228-3235 ◽  
Author(s):  
Rajendra S. Kadam ◽  
Robert. I. Scheinman ◽  
Uday B. Kompella
Keyword(s):  
Cell Line ◽  
Mdck Cell ◽  
In Vitro Model ◽  
Blood Retinal Barrier ◽  
Mdck Cell Line ◽  
Vitro Model

scholarly journals Expanding the Efflux In Vitro Assay Toolbox: A CRISPR-Cas9 Edited MDCK Cell Line with Human BCRP and Completely Lacking Canine MDR1

2021 ◽  
Vol 110 (1) ◽  
pp. 388-396 ◽  
Author(s):  
Christine Wegler ◽  
Meryem Gazit ◽  
Karolina Issa ◽  
Sujay Subramaniam ◽  
Per Artursson ◽  
...  
Keyword(s):  
Cell Line ◽  
Mdck Cell ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Mdck Cell Line

scholarly journals Physical and Functional Interaction of Growth Hormone and Insulin-Like Growth Factor-I Signaling Elements

2004 ◽  
Vol 18 (6) ◽  
pp. 1471-1485 ◽  
Author(s):  
Yao Huang ◽  
Sung-Oh Kim ◽  
Ning Yang ◽  
Jing Jiang ◽  
Stuart J. Frank
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Janus Kinase ◽  
Intact Cells ◽  
Igf I

Abstract GH and IGF-I are critical regulators of growth and metabolism. GH interacts with the GH receptor (GHR), a cytokine superfamily receptor, to activate the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), and initiate intracellular signaling cascades. IGF-I, produced in part in response to GH, binds to the heterotetrameric IGF-I receptor (IGF-IR), which is an intrinsic tyrosine kinase growth factor receptor that triggers proliferation, antiapoptosis, and other biological actions. Previous in vitro and overexpression studies have suggested that JAKs may interact with IGF-IR and that IGF-I stimulation may activate JAKs. In this study, we explore interactions between GHR-JAK2 and IGF-IR signaling pathway elements utilizing the GH and IGF-I-responsive 3T3-F442A and 3T3-L1 preadipocyte cell lines, which endogenously express both the GHR and IGF-IR. We find that GH induces formation of a complex that includes GHR, JAK2, and IGF-IR in these preadipocytes. The assembly of this complex in intact cells is rapid, GH concentration dependent, and can be prevented by a GH antagonist, G120K. However, it is not inhibited by the kinase inhibitor, staurosporine, which markedly inhibits GHR tyrosine phosphorylation. Moreover, complex formation does not appear dependent on GH-induced activation of the ERK or phosphatidylinositol 3-kinase signaling pathways or on the tyrosine phosphorylation of GHR, JAK2, or IGF-IR. These results suggest that GH-induced formation of the GHR-JAK2-IGF-IR complex is governed instead by GH-dependent conformational change(s) in the GHR and/or JAK2. We further demonstrate that GH and IGF-I can synergize in acute aspects of signaling and that IGF-I enhances GH-induced assembly of conformationally active GHRs. These findings suggest the existence of previously unappreciated relationships between these two hormones.

scholarly journals Endogenous and exogenous Na-K-Cl cotransporter expression in a low K-resistant mutant MDCK cell line

2001 ◽  
Vol 280 (6) ◽  
pp. C1607-C1615 ◽  
Author(s):  
John A. Payne ◽  
Christina Ferrell ◽  
Chee Yeun Chung
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Mdck Cell ◽  
Mdck Cells ◽  
Expression System ◽  
Basolateral Membrane ◽  
Resistant Mutant ◽  
Secretory Cells ◽  
Mdck Cell Line ◽  
Low K

A low K-resistant mutant Madin-Darby canine kidney (MDCK) cell line, LK-C1, has been shown previously to lack functional Na-K-Cl cotransporter (NKCC) activity, indicating that it may be a useful NKCC “knockout” cell line for structure-function studies. Using immunological probes, we first characterized the defect in the endogenous NKCC protein of the LK-C1 cells and then fully restored NKCC activity in these cells by stably expressing the human secretory NKCC1 protein (hNKCC1). The endogenous NKCC protein of the LK-C1 cells was expressed at significantly lower levels than in wild-type MDCK cells and was not properly glycosylated. This latter finding indicated that the lack of functional NKCC activity in the LK-C1 cells may be due to the inability to process the protein to the plasma membrane. In contrast, exogenously expressed hNKCC1 protein was properly processed and fully functional at the plasma membrane. Significantly, the exogenous hNKCC1 protein was regulated in a manner similar to the protein in native secretory cells as it was robustly activated by cell shrinkage, calyculin A, and low-Cl incubation. Furthermore, when the LK-C1 cells formed an epithelium on permeable supports, the exogenous hNKCC1 protein was properly polarized and functional at the basolateral membrane. The low levels of endogenous NKCC protein expression, the absence of any endogenous NKCC transport activity, and the ability to form a polarized epithelium indicate that the LK-C1 cells offer an excellent expression system with which to study the molecular physiology of the cation Cl cotransporters.

In-vitro validation of self designed siRNA targeting non-structural 1 gene of Influenza A virus

2015 ◽  
Vol 4 (6) ◽  
pp. 315-322
Author(s):  
Bhawana Jain ◽  
Amita Jain ◽  
Om Prakash ◽  
Ajay K. Singh ◽  
Tanushree Dangi ◽  
...  
Keyword(s):  
Cell Line ◽  
Influenza A Virus ◽  
Mdck Cell ◽  
Influenza A ◽  
Screening Method ◽  
Viral Gene ◽  
Mdck Cell Line ◽  
Ns1 Gene ◽  
Conserved Region

  The genomic variability makes Influenza A virus (IAV) difficult to be con-trolled by existing vaccines or anti-influenza drugs. Viral gene targeting siRNA induces the RNAi mechanism in the host and silents the gene by cleaving mRNA. Objective was to develop siRNA targeting non-structural 1 gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence align-ment) of NS1 gene of IAV strains. To choose the most efficient siRNA, three levels screening method was developed. Ultimately one siRNA duplex was selected on the basis of its unique position in conserved region. siRNA effica-cy was confirmed in vitro on commonly used Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/2011(H3N2)] and Influenza A/pdmH1N1 [A/India/LKO2151/2012(H1N1)]. Of total 173 strains worldwide and 30 strains from India, 32 bp long (position 561 - 592) conserved region was identified. The longest ORF of NS1 gene was targeted by the selected siRNA, which showed 65.5% inhibition in replication of Influenza A/pdmH1N1 and 67.2% inhibition in replication of Influenza A/H3N2 at 48 hpi on MDCK cell line. This study shows that siRNA targeting NS1 may be quite effective in controlling IAV rep-lication so can be used as anti-IAV therapeutic agent.

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