91390576
Download
1 / 31

銘傳大學生物科技學系 姓名 : 尹馭群 學號 :91390576 - PowerPoint PPT Presentation


  • 82 Views
  • Uploaded on

TSLC(TUMOR SUPPRESSOR IN LUNG CANCER)1 SUPPRESSES EPITHELIAL CELL SCATTERING AND TUBULOGENESIS Mari Masuda, et. al. (2005) J. Biol. Chem. 280, 42164-42171. 銘傳大學生物科技學系 姓名 : 尹馭群 學號 :91390576. Introduction. TSLC1 EMT Cyst Cell adhesion molecules Epithelial tissue and connective tissue

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' 銘傳大學生物科技學系 姓名 : 尹馭群 學號 :91390576' - delano


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
91390576

TSLC(TUMOR SUPPRESSOR IN LUNG CANCER)1 SUPPRESSES EPITHELIAL CELL SCATTERING AND TUBULOGENESISMari Masuda, et. al. (2005)J. Biol. Chem. 280, 42164-42171

銘傳大學生物科技學系

姓名: 尹馭群

學號:91390576


Introduction
Introduction CELL SCATTERING AND TUBULOGENESIS

  • TSLC1

  • EMT

  • Cyst

  • Cell adhesion molecules

  • Epithelial tissue and connective tissue

  • ZO-1

  • Tet-off System

  • Cell scattering and tubulogenesis

  • Rac, Rho, Actin cytoskeleton


Tslc1
TSLC1 CELL SCATTERING AND TUBULOGENESIS

  • TSLC/IGSF4A is a tumor suppressor gene in nonsmall cell lung cancer (NSCLC), which encodes a single membrane-spanning glycoprotein belong to the family of immunoglobulin superfamily cell adhesion molecules(IgCAMs)

  • The cytoplasmic tail of TSLC1 contains a juxtamembrane sequence interacting with protein 4.1 and a class II PDZ binding motif

  • In a primary NSCLC we have found a 2-bp deletion in the TSLC1 the cause replacement of the c-terminal 19 amino acid residues, indicating that the cytoplasmic domain of TSLC is crucial for tumor suppression


Epithelial tissue
Epithelial tissue CELL SCATTERING AND TUBULOGENESIS

  • Epithelial tissue covers the whole surface of the body. It is made up of cells closely packed and ranged in one or more layers.

  • Epithelial tissue, regardless of the type, is usually separated from the underlying tissue by a thin sheet of connective tissue; basement membrane. The basement membrane provides structural support for the epithelium and also binds it to neighbouring structures


Connective tissue
Connective tissue CELL SCATTERING AND TUBULOGENESIS

  • Connective tissue is any type of biological tissue with an extensive extracellular matrix and often serves to support, bind together, and protect organs

  • Connective tissue has abundant extracellular matrix

  • Composed by smaller cells and low densities


Emt epithelial mesenchymal transitions
EMT (epithelial mesenchymal transitions) CELL SCATTERING AND TUBULOGENESIS

Epithelial cells are the cells that line virtually every organ in your body and 85% of cancers derive from epithelial cells. During embryonic development epithelial cells sometimes dissolve their junctions with their neighbors and become mesenchymal. Mesenchymal cells have a less rigid shape and are more likely to be motile. Epithelial to mesenchymal transitions, as well as the reverse process, are extremely important for normal development. In addition, these transitions are important in wound healing, and tumor cells that develop from epithelial cells must transform into motile cells in order to metastasize.


Cyst CELL SCATTERING AND TUBULOGENESIS

  • The cyst is a spherical monolayer of polarized cells that encloses a central lummen


Cell adhesion molecules cam
Cell adhesion molecules (CAM) CELL SCATTERING AND TUBULOGENESIS


ZO - 1 CELL SCATTERING AND TUBULOGENESIS

  • A marker of tight junctions, which is normally detected on the apical surfaces facing inwards of parental MDCK cyst

Apical

Basal


Tet off system
Tet-off system CELL SCATTERING AND TUBULOGENESIS


Cell scattering
Cell scattering CELL SCATTERING AND TUBULOGENESIS

  • Cell scattering is used to describe the dispersion of compact colonies of epithelial cells induced by certain soluble factors such as growth factors, cytokines, and phorbol esters

  • Procedures:

    • Cell spreading

    • Dissociation cells from each other

    • Migration as individual cells


Tubulogenesis
Tubulogenesis CELL SCATTERING AND TUBULOGENESIS

  • Tubulogenesis processed during HGF-induced EMT. Generally speaking, it is related with tumor cells’ invasion and matastasis

  • Procedures:

    • Formation of extensions

    • Formation of single file chains

    • Conversion to multilayered cords

    • Maturation of tubules


*Actin cytoskeleton is regulated by small GTP-binding proteins

(Rho, Rac )

  • Previous studies fromothers have shown that a constitutively active Rac mutant abolished HGF-mediated scattering

  • And high in Rac and low in Rho activities are correlated with an epithelial phenotype by stabilizing E-cadherin cell-cell adhesion

  • A transient decrease in Rac activity is essential to induce disassembly of cell-cell junctions


Material and method
Material and Method proteins

  • Cells and proliferation Assay

  • Antibodies

  • Expression Vectors and Transfection

  • Protein Analysis

  • Three-dimensional Culture in Collagen Gels and Tubulogenesis Assay

  • Cell Scattering Assay

  • Immunostaining and Confocal Microscopy

  • Rac and Rho Activation Assay (GST pull-down assay)

  • Statistics


Three dimensional culture
Three-dimensional culture proteins

  • Three-dimensional culture is a powerful tool for investigating the molecular signals, that specify epithelial architecture


Immunostaining
Immunostaining proteins

  • Direct immunofluorescence

  • Indirect immunofluorescence


Result
Result proteins

1.The obtained clones express the transgenes at a nearly equivalent level

2. MDCK Tet-off cells express a low level of endogenous TSLC detected as faint bands

Overexpression of TSLC or any of the truncation mutants does not alter cell growth in MDCK cell


MDCK(-) proteins

TSLC1

ΔC-HA

TSLC1

TSLC1

ZO-1

Merged


Δ4.1-BM proteins

ΔPDZ-BM

TSLC1

TSLC1

ZO-1

Merged

TSLC1


MDCK(-) proteins

GFP

TSLC1

ΔC-HA

HGF(-)

DOX(-)

HGF(+)

DOX(-)

DOX(+)


HGF(-) proteins

DOX(-)

HGF(+)

DOX(-)

DOX(+)

TSLC1

Δ4.1-BM

ΔPDZ-BM


  • Quantitativeanalysis of 30 cysts for each cell clone demonstrated that the differencein tubulogenesis between the Dox-treated (TSLC1 off) and untreated(TSLC1 on) MDCK/TSLC1 cells was statistically significant (p < 0.0001), whereas other cell clones showed no statistically significant difference between the Dox-treated and untreated cysts (p>0.05)


MDCK(-) demonstrated that the differencein tubulogenesis between the Dox-treated (TSLC1 off) and untreated(TSLC1 on) MDCK/TSLC1 cells was statistically significant (

GFP

TSLC1

ΔC-HA

HGF(-)

DOX(-)

HGF(+)

DOX(-)

DOX(+)


HGF(-) demonstrated that the differencein tubulogenesis between the Dox-treated (TSLC1 off) and untreated(TSLC1 on) MDCK/TSLC1 cells was statistically significant (

DOX(-)

HGF(+)

DOX(-)

DOX(+)

TSLC1

Δ4.1-BM

ΔPDZ-BM


A quantitative analysis of cell scattering confirmed that the difference between the Dox-treated and untreated MDCK/TSLC1 cells was statistically significant with p <0.0001. In contrast, other cell clones showed no significant difference between the Dox-treated and untreated cells (p>0.05)


  • the HGF treatedMDCK/TSLC1 cells exhibited prolonged Rac activation, whichwas still observed 4 h after stimulation, but there was no increase in Rho activity.


Conclusion
Conclusion early increase in Rac activity,which returned to basal levels within 15 min as well, as sustained Rho activation

  • Both the Protein 4.1-BM and the PDZ-BM are Necessary for the lateral localization of TSLC1 in cyst grown in a 3D culture

  • Expression of TSLC1 in MDCK cells suppressed HGF-induced tubulogenesis

  • TSLC1 inhibits HGF-induced cell scattering by suppressing induction of EMT

  • MDCK/TSLC1 cells retained E-cadherin-based cell-cell adhesion even after treated with HGF


Conclusion1
Conclusion early increase in Rac activity,which returned to basal levels within 15 min as well, as sustained Rho activation

  • The expression of TSLC1 induces the prolonged activation of Rac and the reduced activation of Rho in HGF-stimulated MDCK cells

  • The regulatory effect of TSLC1 on Rac activity appeared to depend upon its cytoplasmic domain because MDCK/C-HA cells showed a profile of Rac activation panel) similar to that in parental MDCK cells

  • TSLC1 suppresses induction of EMT by modulating the activities of Rac and Rho


The End early increase in Rac activity,which returned to basal levels within 15 min as well, as sustained Rho activation


Conclusion2
Conclusion early increase in Rac activity,which returned to basal levels within 15 min as well, as sustained Rho activation

  • Both the Protein 4.1-BM and the PDZ-BM are Necessary for the lateral localization of TSLC1 in cyst grown in a 3D culture

  • Expression of TSLC1 in MDCK cells suppressed HGF-induced tubulogenesis

  • TSLC1 inhibits HGF-induced cell scattering by suppressing induction of EMT

  • MDCK/TSLC1 cells retained E-cadherin-based cell-cell adhesion even after treated with HGF


ad