Transcript DOX(+)

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
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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
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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
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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 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
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EMT (epithelial mesenchymal transitions)
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
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The cyst is a spherical monolayer of
polarized cells that encloses a central
lummen
Cell adhesion molecules (CAM)
ZO - 1
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A marker of tight junctions, which is
normally detected on the apical surfaces
facing inwards of parental MDCK cyst
Apical
Basal
Tet-off system
Cell scattering
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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:
1. Cell spreading
2. Dissociation cells from each other
3. Migration as individual cells
Tubulogenesis
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Tubulogenesis processed during HGFinduced EMT. Generally speaking, it is
related with tumor cells’ invasion and
matastasis
Procedures:
C.
Formation of extensions
Formation of single file chains
Conversion to multilayered cords
D.
Maturation of tubules
A.
B.
*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
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Material and Method
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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
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Three-dimensional culture is a powerful
tool for investigating the molecular signals,
that specify epithelial architecture
Immunostaining
Direct immunofluorescence
 Indirect immunofluorescence
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Result
1.The obtained clones express the transgenes
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
at
TSLC1
MDCK(-)
TSLC1
ΔC-HA
TSLC1
ZO-1
Merged
TSLC1
TSLC1
Δ4.1-BM
ΔPDZ-BM
TSLC1
ZO-1
Merged
HGF(-)
DOX(-)
MDCK(-)
GFP
TSLC1
ΔC-HA
HGF(+)
DOX(-)
DOX(+)
HGF(-)
DOX(-)
TSLC1
Δ4.1-BM
ΔPDZ-BM
HGF(+)
DOX(-)
DOX(+)
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 Doxtreated and untreated cysts (p>0.05)
HGF(-)
DOX(-)
MDCK(-)
GFP
TSLC1
ΔC-HA
HGF(+)
DOX(-)
DOX(+)
HGF(-)
DOX(-)
TSLC1
Δ4.1-BM
ΔPDZ-BM
HGF(+)
DOX(-)
DOX(+)
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 Doxtreated and untreated cells (p>0.05)
 In response to HGF, the
parental cells showed
immediate early increase
in Rac activity,which
returned to basal levels
within 15 min as well, as
sustained Rho activation
 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
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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
Conclusion
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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
Conclusion
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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