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Craig D. Woodworth, Evan Michael, Laura Smith, and Matthias Nees. Department of Biology, Clarkson
University, Potsdam, NY, USA, and Department of Pediatric Oncology, Hematology & Immunology, University of Heidelberg, Heidelberg, Germany
Inhibition of Epidermal Growth Factor Receptor Function in Cervical Carcinoma
Cells Alters Expression of Genes Involved in Invasion, Apoptosis, Inflammation, and Cell
Cycle Regulation
EGF-RErbB-2ErbB-3ErbB-4
EGFTGF-HB-EGF
-cellulinepiregulinamphiregulin
tyrosine kinase domain links to signaling pathways
The Epidermal Growth Factor Receptor (EGF-R) is a Membrane Tyrosine Kinase
proliferation motility angiogenesis
differentiation apoptosis (cell death)
EGF
tyrosinephosphorylation
EGF-RErbB-2ErbB-3ErbB-4
P P
TGF-HB-EGF
-cellulinepiregulinamphiregulin
Binding of EGF Induces Dimerization, Tyrosine Phosphorylation and Signaling
• HPV-16 E6 and E5 genes stimulate expression and activation of the epidermal growth factor receptor (EGF-R), respectively
• Expression of the EGF-R is increased in papillomas and cancers of the uterine cervix, and patients with the highest EGF-R expression often have a poor prognosis
• Targeted disruption of the EGF-R gene in a mouse model inhibits formation of papillomas and carcinomas from HPV-immortalized keratinocytes
The EGF-R as a Cofactorfor HPV-Associated Cancer
4-[(3-Bromophenyl)amino]-6,7-imethoxyquinazoline a potent and specific inhibitor of the tyrosine kinase
activity of the EGF-R (IC50 = 25pM)
Does Inhibition of EGF-R Function Alter Growth, Differentiation, or Gene Expression
of Cervical Carcinoma Cells?
PD 153035
0
CXT3CXT2
3.01.00.30.1
EGF-R
P-Tyr
EGF-R
P-Tyr
0 3.01.00.30.1M M
PD153035 Inhibits Tyrosine Phosphorylationof the EGF-R in a Dose-Dependent Manner
Construct rafts composed of collagen and fibroblasts
Allow fibroblasts to contract raft for 2 days
Raise rafts on steel mesh grids and maintain at the air-liquid interface
Add normal human cervical cells or cervical cancer cells to the surface of raft
After 10 days scrape epithelia from raft and purify RNA, or fix the raft for histology
Organotypic Culture to Promote Cell-Cell and Cell-Matrix Interactions
Normal cervical cells
CXT2 carcinoma cells
Carcinoma Cells Form Dysplastic Epitheliaand Invade the Underlying Collagen
untreated
0.3 M
3.0 M
EGF-R Inhibitor PD153035 Blocks Invasion
Tumor 1 Tumor 2 Tumor 3
Cel
ls i
nva
din
g g
el
0
20
40
60
80
100
3.0 M
0.3 Muntreated
EGF-R Inhibitor PD153035 Blocks Invasionin a Dose-Dependent Manner
microarray protocol microarray results
Identification of Genes DifferentiallyExpressed After PD153035 Treatment
Inhibition of the EGF-R Alters Expressionof Several Clusters of Genes
Immune response
attachment and motility
cell cycle
inflammation
increased
decreased
symbol gene identification and description
ITGA8 integrin alpha 8, cell-cell interactions
ITGAX integrin alpha X, similar to alpha integrins
CTNND2 catenin, cadherin associated protein
ITGB1 integrin beta 1, fibronectin receptor
SELE selectin E endothelial adhesion molecule
DDR2 discoidin, required for cell adhesionACTN1 alpha 1 actininMMP1 matrix metalloproteinase 1 (collagenase)
PD153035 Alters Expression of Genes that Regulate Attachment and Motility
symbol gene identification and description
XCL1 chemokine ligand 1, attracts leukocytes
CX3CL1 fractalkine, chemotactic for T cells
CCL3 MIP-1inflammatory and chemotactic
CXCR6 chemokine receptor 6, G protein receptor
IL1R2 IL1 receptor type II, decoy receptor
IL-6 interleukin 6, proinflammatory cytokine
IL-7 Interleukin 7, hematopoietic growth factor
IRF5 interferon regulatory factor 5
TNFSF4 member of tumor necrosis factor family
PD153035 Increases Expression of RNAs for Cytokines and Chemokines
Verification of Selected Microarray Results Using Real Time RT-PCR
0
1
2
3
4
5
CXCR6
CX3CL1
CCL3IL
6IL
7
IKB a
lpha
DAPK1
CCL3
RELA
IL1
beta
NFKBIA
CCL11
IL1
alpha
Fo
ld in
crea
se
No treatment0.1 M PD1530350.3 M PD153035
• Cervical cancer cells produce dysplastic epithelia and invade the underlying collagen in organotypic culture
• Inhibition of EGF-R tyrosine kinase activity by PD153035 decreases invasion in a dose-dependent manner
• Inhibition of the EGF-R up regulates expression of genes that mediate attachment and inflammation, and down regulates many genes that stimulate growth
Summary
Matthias Nees University of Heidelberg
Evan Michael University of Michigan
Laura Smith Clarkson University Sarah Allen Mandy Heitzke April Krumnow
Acknowledgements