Antisense studies on base excision repair genes and their effects on cellular sensitivity to ionizing radiation [Elektronische Ressource] / vorgelegt von Sebastian Hausmann

Chris Dulabone

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2011
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

INAUGURAL-DISSERTATION

zur Erlangung der Doktorwürde
der
Naturwissenschaftlich-Mathematischen Gesamtfakultä t
der
Ruprecht-Karls-Universität
Heidelberg






vorgelegt von
Apotheker Sebastian Hausmann
geboren in Northeim
Tag der mündlichen Prüfung:



Antisense studies on base excision repair genes atnhdeir effects
on cellular sensitivity to ionizing radiation













Referees: PD Dr. Odilia Popanda
Prof. Dr. Gert Fricker



To my family





Division: Epigenomics and Cancer Risk Factors
Head of the division: Prof. Dr. Christoph Plass
German Cancer Research Center (DKFZ), Heidelberg



Declarations according to § 8 (3) b) and c) ofo ctthoer adl degree regulations:

I hereby declare that I have written the subdmissitetretda tion myself and in this process
have used no other sources or materials than tehxopsre ssly indicated,

I hereby declare that I have not applied to ibne de xaat many other institution, nor have
I used the dissertation in this or any other tf oarnmy aother institution as an examination
paper, nor submitted it to any other facultyi sases rta tdion.



Heidelberg, February 15, 2011 Sebastian Hauns man


Table of contents

Summary ............................................................. ................ x
Zusammenfassung ....................................................... ............ xii
Abbreviations ............................................................................................... ........................ xiv
1 Introduction ....................................................... .............. 1
1.1 Breast cancer .................................................. ......... 1........
1.1.1 Breast cancer risk factors .................................... .. 1.....................
1.2 Treatment of cancer ....................................................................... ........... 2.............
1.2.1 Surgery ..................................................... ....... 3...........
1.2.2 Chemotherapy ............................................... ... 3..................
1.2.3 Radiotherapy ............................................... ..... 3................
1.3 Side effects of radiotherapy .......................................................... ......... 4................
1.4 Cellular response to ionizing radiation .............................. .. 4.....................
1.5 Repair mechanisms .............................................. ...... 5.............
1.5.1 Double-strand break repair .................................... 6........................
1.5.1.1 Non-homologous end joining ............................... ..................... 7
1.5.1.2 Homologous recombination ................................................ .................... 8
1.5.2 Nucleotide excision repair ...................................... . 8......................
1.5.3 Base excision repair .......................................... ... 9..................
1.5.3. 1 Polymorphisms in BER genes and radiosensiti.vi.ty. .............. ............. 12
1.5.3. 2 APEX nuclease (multifunctional DNA repair enzym .e.). .1.......... ........... 14
1.5.3. 3 X-ray repair cross complementing group 1 .................... ............... 16
1.6 Expression of BER enzymes and radiosensit.iv.i.ty. .................... ...................... 18
1.7 Background of this thesis ........................................ .... 1.9...............
1.8 Aims of the study ........................................................................... ........... 2.0...........
2 Materials and Methods ............................................... ...... 2.1..........
2.1 Materials ....................................................... ........ 2.1......
2.1.1 Cell culture ................................................. ..... 2.1.............
2.1.2 Cell culture media and supplements ............................. ..................... 21
2.1.3 Cell lines .................................................................................. ........... 2.1...........
2.1.4 Centrifuges.................................................. .... 2.1...............
2.1.5 Electrophoresis ............................................. ... 2.2.................
2.1.6 Enzymes ................................................... .... 2.2..............
2.1.7 Materials ................................................................................. ......... 2.2.............
2.1.8 Nucleic acids ............................................... .... 2.3...............
2.1.9 Other devices ........................................................................... ..... 2.4................
2.1.10 PCR devices ................................................ .... 2.4...............
2.1.11 Solutions, chemicals, and buffers .............................5. ....................... 2
2.1.12 Ready to use kits and solutions .............................2.6. ........................
2.1.13 Antibodies ................................................................................ ....... 2.7..............
2.1.14 Software and databases .................................... 2.7 ........................
2.2 Methods ....................................................... ........ 2.8.......
2.2.1 Description of cell lines, their cultivation, arynodc ocnservation .......... ....... 28
2.2.1.1 MCF7 ................................................................................ . 2.8....................
2.2.1.2 HMEpC .................................................. 2.8.....................
2.2.1.3 Determination of cell viability .............................. ..................... 29
2.2.1.4 Irradiation of the cells .................................2.9. ........................
2.2.2 RNA interference ...................................................................... . 3.0...................
2.2.2.1 Optimization of transfection conditions ....................... ................. 30
2.2.2.2 Transfection procedure .................................... ...................... 31
2.2.3 RNA isolation and analysis .................................... 3. 1.......................
2.2.3.1 Qualification and quantification of the isolateAd .R..N............ ............. 32
2.2.3.2 First-strand cDNA Synthesis .............................................. ..................... 32
2.2.3.3 Verification of the cDNA integrity .......................... .................... 33
2.2.3.4 Quantitative real-time RT-PCR ............................. .................... 34
2.2.4 Gene expression analysis on Illumina BeadArra.y.s. ................. ................. 37
2.2.5 Protein analysis using Western blot ........................................ ...................... 38
2.2.5.1 SDS polyacrylamide gel electrophoresis ....................... ................ 39
2.2.5.2 Blotting and immunodetection .............................. .................. 39
2.2.6 Single cell gel electrophoresis ................................ 4. 0........................
2.2.7 Clonogenic assay of ceilnls vitro ............................................. ............. 4.2........
2.2.8 Sulforhodamine B assay ..................................... 4. 3........................
2.2.8.1 Radiation treatment ...................................... ....................... 44
2.2.8.2 Temozolomide treatment .................................. .................... 44
2.2.9 Measurement of H2AX ......................................... ............... 45
2.2.9.1 Immunofluorescence microscopy ......................................... ................. 45
2.2.9.2 Measurement of H2AX and cell cycle analysis after irradiationlo wby f
cytometry ............................................... 4.6.....................
2.3 Statistical methods .............................................. ..... 4.7............
2.3.1 Mean, standard deviation, standard error of the nm, emaedian .......... ....... 47
gg
2.3.2 t-statistics .................................................. ..... 4.7............
2.3.3 Evaluation of microarrays ...................................................... 4. 7.......................
3 Results........................................................... ............ 49
3.1 Silencing of the DNA repair genAePsE X1 andX RCC1 ...................... .................... 49
3.1.1 Optimized transfection conditions lead to effec tkivneockdown oAfP EX1
mRNA transcripts in MCF7 and HMEpC cells .............................. .................. 50
3.1.2 Verification of RNA quality .................................. 5. 3.......................
3.1.3 Verification of cDNA integrity after first-stryannthde sis ............... ............. 53
3.2 Investigations in MCF7 cells ....................................... ... 5.5................
3.2.1 Effective knockdownA oPfE X1 72 h after treatment with siRNA .....5.5. ..........
3.2.2 XRCC1 expression in MCF7 is down-regulated after tracntsiofne with 100 nM
XRCC1 siRNA ............................................... 5.6......................
3.2.3 Decreased mRNA and protei