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Modulation of Cytokine Gene
Expression by EGF-R Inhibition
Craig Woodworth, Department of Biology,
Clarkson University
Background
• HPV-16 E6 and E5 genes stimulate expression
and activation of the EGF-R, respectively
• Expression of the EGF-R is strongly increased
in papillomas and cancers of the uterine cervix
• Patients with the highest EGF-R expression
often have a poor prognosis
• Targeted disruption of the EGF-R gene inhibits
formation of papillomas and carcinomas in a
mouse model
Does PD153035 inhibit immortalization of
keratinocytes or expression of cytokines?
PD 153035
4-[(3-Bromophenyl)amino]-6,7-dimethoxyquinazoline
an extremely potent and specific inhibitor of the tyrosine kinase
activity of the EGF-R (IC50 = 25pM)
PD153035 inhibits tyrosine phosphorylation
of the EGF-R in a dose-dependent manner
CXT2
0
CXT3
0.1 0.3 1.0 3.0 mM
0
P-Tyr
P-Tyr
EGF-R
EGF-R
0.1 0.3 1.0 3.0 mM
EGF-R inhibitor PD153035 inhibits growth of
cervical carcinoma cells in monolayer culture
PD153035
EGF-R
tumor-1
100
Percent growth
AG1295
PDGF-R
100
tumor-2
tumor-3
75
tumor-4
75
50
50
25
25
0
0
0
0.03
.01
0.3
Conc. (mM)
1
3
0
0.1
0.3
1
Conc. (mM)
3
10
EGF-R Inhibition blocks
immortalization by HPV-16 E6/E7
untreated
0.1 mM PD153035
1.0 mM PD153035
160
Colonies / dish
140
120
100
80
60
40
20
0
Exp1
Exp2
Exp3
Organotypic culture of cervical cells
results in stratified squamous epithelia
Construct rafts composed of collagen
and fibroblasts
Allow fibroblasts to contract raft for 2
days
Add normal human cervical cells or
cervical cancer cells to the surface of raft
Raise rafts on steel mesh grids and
maintain at the air-liquid interface
After 10 days scrape epithelia from raft
and purify RNA, or fix the raft for
histology
Carcinoma cells form dysplastic epithelia
and invade the underlying collagen
Normal cervical cells
CXT2 carcinoma cells
EGF-R inhibitor PD153035 blocks invasion
untreated
0.3 mM
3.0 mM
EGF-R inhibitor PD153035 inhibits invasion
of carcinoma cells into collagen rafts
Cells invading gel
100
untreated
0.3 mM
3.0 mM
80
60
40
20
0
Tumor 1
Tumor 2
Tumor 3
PD153035 induces
proinflammatory cytokines
PD153035 alters expression of genes
that regulate apoptosis
PD153035 alters expression
of cell cycle genes
Summary
• Inhibition of EGF-R function by PD153035
blocks immortalization of human cervical cells
by HPV-16 E6/E7 genes
• 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 dosedependent manner
• Inhibition of EGF-R activity stimulates multiple
genes that mediate inflammation
Questions
• Could EGF-R inhibitors reduce the incidence
of HPV infection or dysplasia?
• Does inhibition of EGF-R function stimulate
inflammation or innate immune responses?
• The keratinocyte; NF-kB activation,
expression of HLA class I genes, production
of cytokines, HPV-16 gene expression
Acknowledgements
Darci Gaiotti
Evan Michael
Joel Geoghegan
Donald Vice
Eudora Jones
Peter Munson
Laura Hansen
Stuart Yuspa
Matthias Nees