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銘傳大學生物科技學系 姓名 : 尹馭群 學號 :91390576

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

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銘傳大學生物科技學系 姓名 : 尹馭群 學號 :91390576

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  1. 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

  2. Introduction • TSLC1 • EMT • Cyst • Cell adhesion molecules • Epithelial tissue and connective tissue • ZO-1 • Tet-off System • Cell scattering and tubulogenesis • Rac, Rho, Actin cytoskeleton

  3. TSLC1 • 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

  4. Epithelial tissue • 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

  5. 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

  6. 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.

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

  8. Cell adhesion molecules (CAM)

  9. ZO - 1 • A marker of tight junctions, which is normally detected on the apical surfaces facing inwards of parental MDCK cyst Apical Basal

  10. Tet-off system

  11. Cell scattering • 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

  12. 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

  13. *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

  14. Material and Method • 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

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

  16. Immunostaining • Direct immunofluorescence • Indirect immunofluorescence

  17. Result 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

  18. MDCK(-) TSLC1 ΔC-HA TSLC1 TSLC1 ZO-1 Merged

  19. Δ4.1-BM ΔPDZ-BM TSLC1 TSLC1 ZO-1 Merged TSLC1

  20. MDCK(-) GFP TSLC1 ΔC-HA HGF(-) DOX(-) HGF(+) DOX(-) DOX(+)

  21. HGF(-) DOX(-) HGF(+) DOX(-) DOX(+) TSLC1 Δ4.1-BM ΔPDZ-BM

  22. 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)

  23. MDCK(-) GFP TSLC1 ΔC-HA HGF(-) DOX(-) HGF(+) DOX(-) DOX(+)

  24. HGF(-) DOX(-) HGF(+) DOX(-) DOX(+) TSLC1 Δ4.1-BM ΔPDZ-BM

  25. 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)

  26. 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.

  27. Conclusion • 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

  28. Conclusion • 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

  29. The End

  30. Conclusion • 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

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