Predicting the response of cervical cancer to radiotherapy
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Transcript of Predicting the response of cervical cancer to radiotherapy
![Page 1: Predicting the response of cervical cancer to radiotherapy](https://reader035.fdocuments.in/reader035/viewer/2022070417/568152b4550346895dc0d859/html5/thumbnails/1.jpg)
Department of Gynecologic oncology
Frank Roossink
Identification of hypermethylation based
markers
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• The second most common malignancy among women world-wide. In the Netherlands 700 patients present with cervical cancer
each year. World-wide this number is 450,000 cases and 250,000 deaths.
• Disproportionally towards less developed countries.
21-04-23 Identification of RT response methylation markers in cervical cancer 2
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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Data from Globocan (2002)Data from Globocan (2002)
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
![Page 4: Predicting the response of cervical cancer to radiotherapy](https://reader035.fdocuments.in/reader035/viewer/2022070417/568152b4550346895dc0d859/html5/thumbnails/4.jpg)
• The second most common malignancy among women world-wide. In the Netherlands 700 patients present with cervical cancer
each year. World-wide this number is 450,000 cases and 250,000 deaths.
• Disproportionally towards less developed countries.
• 99.7% of all cervical cancer patients are human papilloma virus(HPV) positive. (Waalbomers, 1999)
70% is due to infection with types HPV-16 and HPV-18.
21-04-23 Identification of RT response methylation markers in cervical cancer 4
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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21-04-23 Identification of RT response methylation markers in cervical cancer 5
0.3%
0.5%
0.6%
0.7%
1.0%
1.2%
1.3%
1.4%
2.2%
2.3%
2.6%
2.9%
17.3%
53.5%
6.7%
Roden and Wu Roden and Wu Nature ReviewsNature Reviews (2006) (2006)
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• Cancer is a disease of the genes.
• Changes in expression patterns of genes are thought to be the cause for the development of cancer.
• In cervical cancer these changes in expression are thought to initiate with the establishment HPV infection.
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
![Page 7: Predicting the response of cervical cancer to radiotherapy](https://reader035.fdocuments.in/reader035/viewer/2022070417/568152b4550346895dc0d859/html5/thumbnails/7.jpg)
Dermis
Basal layerBasement membrane
Midzone
Superficial zone
Normal cervix Squamous Intraepithelial lesion (SIL)
Low grade High grade
Invasive cancerASCUS
HPV infection
Infectious HPV particles
80% clearance
Viral integration
E6/E7 deregulation
Proliferation
20% Lesions Genetic instability
Adapted from Snijders et alAdapted from Snijders et al. J. of pathology . J. of pathology (2006), Woodman et al. (2006), Woodman et al. Nature reviewsNature reviews (2007). (2007).
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Dermis
Basal layerBasement membrane
Midzone
Superficial zone
Normal cervix Squamous Intraepithelial lesion (SIL)
Low grade High grade
Invasive cancerASCUS
-Loss of Chr. 6p-DR of MHC class I
Adapted from Snijders et alAdapted from Snijders et al. J. of pathology . J. of pathology (2006), Woodman et al. (2006), Woodman et al. Nature reviewsNature reviews (2007). (2007).
-Loss of Chr. 3p,4q, 6q and 10p-UR telomerase
-DR GATA-3
Immortalization
-Loss of Chr. 1-DR TSLC1
Invasion
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• Cancer is a disease of the genes.
• Changes in expression patterns of genes are thought to be the cause to the development of cancer.
• In cervical cancer these changes in expression are thought to initiate with the establishment HPV infection.
• Changes in expression patterns can be due to genetic or epigenetic alterations.
21-04-23 Identification of RT response methylation markers in cervical cancer 10
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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“The branch of biology which studies
the causal interactions between genes and their products which bring the phenotype into being”
C.H. Waddington, 1942
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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Waddington (1948)Waddington (1948)
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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“Heritable states of gene-expression that are not due to
alterations in the underlying DNA sequence itself.”
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
3.5x
DNA packaging:• Every mitotic cell has 23 pair chromosomes.
• Approximately 30,000 genes.• Approximately 2m DNA per cell.
• Each individual consists of 10 billion cells.• That means 500,000 trips around the equator or
3.5 trips from the Sun to Pluto.
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Molecular biology of the cell, 4Molecular biology of the cell, 4thth edition edition
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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30nm fiber“Beads on a string”
Histone tail modificationsDNA double helix
Adapted from: http://www.newscientist.com/data/images/archive/2386/23865001.jpgAdapted from: http://www.newscientist.com/data/images/archive/2386/23865001.jpgMolecular biology of the cell, 4Molecular biology of the cell, 4thth edition edition
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REF!!!REF!!!
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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Histone modifications
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Latham et al. Latham et al. Nature Structural & Molecular Biology, 2007Nature Structural & Molecular Biology, 2007
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• DNA methylation occurs at the 5th position of a cytosine.
• Cytosine that precede Guanine can become methylated So-called CpG dinucleotides.
• Methylation is mediated by DNA methyl transferases (DNMTs) de novo methylation
• DNMT-3a and DNMT-3b Maintenance
• DNMT-1
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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21
Frontal viewSide view
Adapted from O’Gara et al.J Mol Biol. 1996 Sep 6;261(5):634-45
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Frontal view
Adapted from O’Gara et al.J Mol Biol. 1996 Sep 6;261(5):634-45
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• CpG dinucleotides are often found in clusters. So-called CpG islands.
• CpG-island A region with at least 200bp A G-C percentage of >50% CpG-ratio Obs-Exp >0.6 Found in promoter regions of >50% of all genes.
• Promoter hypermethylation can silence genes.
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
Normal Cancer
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To identify genes, which methylation status predicts the
response to chemoradiation.
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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Different methylation pattern in responding patients vs. non-
responding patients.
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• Originally our KWF-application listed 84 genes: 52 genes from array data. 32 genes from literature.
• Together with OMS, we will analyze our patient material using a Methylation Specific PCR based screen.
• Our new and improved list consists of 800+ genes.
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
![Page 27: Predicting the response of cervical cancer to radiotherapy](https://reader035.fdocuments.in/reader035/viewer/2022070417/568152b4550346895dc0d859/html5/thumbnails/27.jpg)
• MSP-based screening tool.• Combines the large screen capabilities of microarray
with the semi-quantitative analysis of Q-PCR.
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• Methylation specific PCR Bisulfite conversion.
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A C G C G C G C C
A C G C G C G U U
Methylated DNA Unmethylated DNA
Differential sequenceDifferential sequence
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• Nucleoside DNMT inhibitors: DAC (Decitabine), 5-aza-C, Zebularin.
• Non-Nucleoside DNMT inhibitors: Hydralazine, EGCG, RG108, Procaine Procainamide
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
Medium co
ntrol
M U M U M U M U M U M U M U
200nM DAC
M U 300nM
TSA
Combi DAC/T
SA 10µM
Hydralazin
e 1mM
VPA
Combi Hyd
r./VPA
5µM DAC
DAPK
M U M U
Leuco
cyte
IV
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
Medium co
ntrol
5µM DAC
200nM DAC
M U M U M U M U M U M U M U M U M U M U M U M U
40µM Hyd
ralazine
1mM Hyd
ralazine
10µM Pro
cainamide
500µM Pro
cainamide
120µM Ze
bularine
1mM Ze
bularine
Medium re
seed
200nM DAC + 300nM
TSA Rese
ed
1mM Ze
bularine re
seed
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• Summary Procainamide is a FDA approved drug which has demethylating
properties.• However, it has severe side-effects.
Also Zebularin has demethylating properties.• Draw-back: it kills monkeys…
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• Validation in a larger cohort of patient samples. Amsterdam series UMCG
• In vitro validation of our candidate genes. Over-expression of our candidate genes
• Clonogenic assay analysis. Knock-down of our candidate genes
• Clonogenic assay
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
Seeding of cells
IrradiationDay 10, 2, 4, 6, 8 and 10Gy
Read-outDay 10
Day 0
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Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
SiHaHeLa
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
SiHa
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
SiHaHeLaCaSki
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
Seeding of cells
Read-outDay 15
Day 0
DAC/TSA treatment
Day 2-4 200nM DACDay 4 300nM TSA
Seeding of cellsDay 5
After 6h, irradiation.0, 2, 4, 6, 8 & 10 Gy
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
SiHa Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
Medium
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
SiHa Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
MediumDAC/TSA (200nM/300nM)
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
SiHa Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
MediumDAC/TSA (200nM/300nM)Zubularine (1mM)
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
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21-04-23 Identification of RT response methylation markers in cervical cancer 38
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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• pcDNA3-HA-TAp73α Gift from G. Melino, Rome Italy
• pIRES2-EGFP-TAp73α
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
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21-04-23 Identification of RT response methylation markers in cervical cancer 40
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
SiHa pcDNA3-HA-TAp73α PBS control SiHa medium control αHA-tag (Y11) 1:50 SiHa pcDNA3-HA-TAp73α αHA-tag (Y11) 1:50
Appr. 37% transfection efficiency
SiH
a un
tran
sfec
ted
SiH
a pc
DN
A3-H
A-TA
p73α
SiH
a un
tran
sfec
ted
SiH
a pc
DN
A3-H
A-TA
p73α
SiH
a un
tran
sfec
ted
SiH
a pc
DN
A3-H
A-TA
p73α
Mar
ker
Mar
ker
Mar
ker
HA-TAp73α
αHA (F7, monoclonal mouse) 1:200 1hour, 30s exposure
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pIRES2-EGFP
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• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
CMV
G.O.I.
IRES
GFP
KanR
NeoR
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• MSP analysis of the selected genes, to see what the methylation status is of our 4 cell lines.
• RT-PCR to see if the genes are transcribed.
• Clone into pIRES2-EGFP.
• Develop RNAi constructs.
• Clonogenic assay of transfected cellswith RNAi and plasmid constructs
21-04-23 Identification of RT response methylation markers in cervical cancer 42
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of
investigation
• Summary
• Acknowledgements
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• HeLa, SiHa are similar sensitive to RT in the clonogenic setting
• CaSki is more resistant than SiHa and HeLa.
• After DAC/TSA and Zebularin treatment prior to RT, SiHa cells are not changed in sensitivity to RT.
• Although procainamide is FDA approved (for arrythmia), we are unlikely to use it due to side-effects.
• Cloning strategies in progress
21-04-23 Identification of RT response methylation markers in cervical cancer 43
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
![Page 43: Predicting the response of cervical cancer to radiotherapy](https://reader035.fdocuments.in/reader035/viewer/2022070417/568152b4550346895dc0d859/html5/thumbnails/43.jpg)
• Our patient-material screen is starting shortly. Our candidate gene list has probably around 800 genes. Test samples already have been sent.
21-04-23 Identification of RT response methylation markers in cervical cancer 44
• Introduction Cervical cancer Epigenetics• Chromatin
structure• DNA methylation
• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of investigation
• Summary
• Acknowledgements
![Page 44: Predicting the response of cervical cancer to radiotherapy](https://reader035.fdocuments.in/reader035/viewer/2022070417/568152b4550346895dc0d859/html5/thumbnails/44.jpg)
21-04-2345
Gynecologic oncology G. Bea A. Wisman Mirjam Kok Maartje G. Noordhuis Ate G.J. van der Zee
Pathology Ed Schuuring
Medical oncology Steven de Jong