modulation of cytokine gene expression by egf-r inhibition
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/1.jpg)
Craig Woodworth, Department of Biology,Clarkson University
Modulation of Cytokine GeneExpression by EGF-R Inhibition
![Page 2: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/2.jpg)
• 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
Background
![Page 3: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/3.jpg)
PD 1530354-[(3-Bromophenyl)amino]-6,7-dimethoxyquinazoline
an extremely potent and specific inhibitor of the tyrosine kinaseactivity of the EGF-R (IC50 = 25pM)
Does PD153035 inhibit immortalization of keratinocytes or expression of cytokines?
![Page 4: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/4.jpg)
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
![Page 5: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/5.jpg)
100
50
25
0
75
31.01 0.30 0.03
tumor-4
tumor-1
tumor-3
tumor-2
Per
cen
t g
row
th
PD153035EGF-R
Conc. (M)
0
25
50
75
100
0 0.1 0.3 1 3 10
AG1295PDGF-R
Conc. (M)
EGF-R inhibitor PD153035 inhibits growth of cervical carcinoma cells in monolayer culture
![Page 6: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/6.jpg)
EGF-R Inhibition blocksimmortalization by HPV-16 E6/E7
0
20
40
60
80
100
120
140
160
Exp1 Exp2 Exp3
Co
lon
ies
/ d
ish
untreated0.1 M PD1530351.0 M PD153035
![Page 7: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/7.jpg)
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 of cervical cells results in stratified squamous epithelia
![Page 8: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/8.jpg)
Normal cervical cells
CXT2 carcinoma cells
Carcinoma cells form dysplastic epitheliaand invade the underlying collagen
![Page 9: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/9.jpg)
untreated
0.3 M
3.0 M
EGF-R inhibitor PD153035 blocks invasion
![Page 10: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/10.jpg)
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 inhibits invasionof carcinoma cells into collagen rafts
![Page 11: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/11.jpg)
PD153035 inducesproinflammatory cytokines
![Page 12: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/12.jpg)
PD153035 alters expression of genes that regulate apoptosis
![Page 13: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/13.jpg)
PD153035 alters expressionof cell cycle genes
![Page 14: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/14.jpg)
• 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 dose-dependent manner
• Inhibition of EGF-R activity stimulates multiple genes that mediate inflammation
Summary
![Page 15: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/15.jpg)
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
![Page 16: Modulation of Cytokine Gene Expression by EGF-R Inhibition](https://reader036.vdocuments.mx/reader036/viewer/2022062809/56815a25550346895dc767a7/html5/thumbnails/16.jpg)
Darci GaiottiEvan MichaelJoel GeogheganDonald ViceEudora Jones
Peter MunsonLaura HansenStuart YuspaMatthias Nees
Acknowledgements