Cet ouvrage et des milliers d'autres font partie de la bibliothèque YouScribe
Obtenez un accès à la bibliothèque pour les lire en ligne
En savoir plus

Partagez cette publication




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 protein levels after simultasn esoiluencing of both
APEX1 andX RCC1 ............................................. .................. 57
3.2.4 Growth characteristics after knockdown of APECXC1,1 , XaRnd DKO ....... ... 59
3.2.5 Analysis of gene expression profiles ............................ ...................... 62
3.2.5. 1 Pathway analysis 24 h after silencingA PoEfX 1, XRCC1, and DKO ...... .. 62
3.2.5. 2 Pathway analysis 48 h after transfection .................... ................. 63
3.2.6 Changes of DNA repair pathways ............................... ...................... 64
3.2.6. 1 Deregulation of DNA repair pathways 24 h aftenr csinileg ........... ........ 65
3.2.6. 2 Gene expression is still altered 48 h after knwocnk .do............. ............ 67
3.2.6. 3 Comparison of differentially expressed DNA repeanire sg ............ ........ 67
3.2.7 General conditions for the irradiation treatme.n.t. .................. .................. 69
3.2.8 Irradiation of silenced and control cells show eddi fnfoerence of radiation
sensitivity .................................................. ...... 6.9...........
3.2.9 Initial DNA damage induction is affected aftdeira tirorna of silenced cells as
measured by the Comet Assay ................................ ....................... 71
3.2.10 Formation of DNA double strand breaks is not eadf faefctetr silencing of
APEX1 andX RCC1 ................................................................... ....................... 73
3.2.11 Cell cycle distribution of cells is affectedtr anfstfeer ction withA PEX1 siRNA
but not after additional treatment with ionaizdiniagt iorn .............. ............ 74
3.2.12 The irradiation of silenced cells results in chsa nig ethe gene expression
patterns ................................................... ..... 7.6............
3.2.12.1 Pathway analysis of radiation-induced gene regounla tiin MCF7 cells
24 h after silencing AoPfE X1 andX RCC1 ........................ ....................... 77
3.2.12.2 Pathway analysis 48 h after silencing and addilt ioirnradiation ...... .... 80
3.2.13 Changes in DNA repair and DNA damage response patyhsw after
irradiation ................................................................................ ......... 8.2............
3.2.13.1 Gene expression profiles 24 h after transfectiodn suabnsequent
irradiation with 5 Gy.....................................................8.2. ........................
3.2.13.2 Small changes in the gene expression patternsl eonfc esdi cells 48 h
after transfection and irradiation with 5 .G.y. ................. ................. 84
3.2.13.3 Comparison of DNA repair pathways obtained 24 a8n dh 4of silencing ..
...................................................... ..... 8.6.............
3.2.14 Challenging BER with temozolomide leads to grnohwitbhi tiion inX RCC1
deficient cells .............................................. ..... 8.6.............
3.3 Investigations in HMEpC ......................................... ... 8.9................
3.3.1 Strong inhibition oAfP EX1 after transfection with 100 AnPM EX1 siRNA ... . 89
3.3.2 XRCC1 mRNA is strongly reduced in its expression ailefntecri nsg of the gene
.......................................................... ........ 9.0......
3.3.3 Double knockdown oAfP EX1 andX RCC1 leads to reduced mRNA and protein
levels of both genes ........................................ .. 9.1..................
3.3.4 Growth characteristics are not influenced afetenrc isnigl ............... ............. 93
3.3.5 Investigations of gene expression patterns aflteenrc isnig ............... ............ 95
3.3.5. 1 Pathway analysis 24 h after silencingA PoEfX 1, XRCC1 and DKO ...... ... 96
3.3.5. 2 Pathway analysis 48 h after silencingA PoEfX 1, XRCC1, and DKO ...... .. 97
3.3.6 Gene expression changes of DNA repair genes .................... ................. 99
3.3.6. 1 XRCC1-silenced cells show multiple changes in their geexnperession
pattern 24 h after transfection ......................................... ..................... 99
3.3.6. 2 Several genes are differentially expressed 48 ehr atrfatnsfection .... . 101
3.3.6. 3 Comparison of changes in the DNA repair pathwa yhs 2a4nd 48 h after
knockdown .......................................... 1.0.1. .......................
3.3.7 Expression levels oAfP EX1 andX RCC1 are strongly reduced at the time of
irradiation in the functional assays ...........................3. ...................... 10
3.3.8 Radiosensitivity of cells is not affected aefntceirn gs ilofA PEX1 andX RCC1
and subsequent treatment with varying doses .o.f. .I.R.. ............. .............. 103
3.3.9 Reduced protein levels of APEX1 and XRCC1 dof luneont cien the initial
damage induction .................................................................. 1. 0.5....................
3.3.10 Formation of H2AX foci is not affected after knockdoAwPnE Xo1f and
XRCC1 ..................................................... .1.0.6..................
3.3.11 Gene expression patterns after transfection andit ioandadl irradiation .... 106
3.3.11.1 Pathway analysis 24 h after transfection and aodndailt iirradiation ... 107
3.3.11.2 Pathway analysis 48 h after transfection and oandadli tiirradiation ... 109
3.3.12 Changes in DNA repair pathways ............................... .................... 111
3.3.12.1 Silencing of target genes in HMEpC and additiroenaatlm tent with IR
causes large changes in the gene expression np atotfe rDNA repair
genes ............................................................................... 1.1.1...................
g
3.3.12.2 Irradiation of silenced cells leads to a dereognu loafti genes of several
DNA repair pathways 48 h after transfectio.n. ....................... ............. 113
3.3.12.3 Comparison of DNA repair pathways obtained 24 a8n dh 4after
transfection and additional treatment with .I.R.. ............... ............. 115
4 Discussion ......................................................... ......... 1.1.6.
4.1 MCF7 .............................................................................................. ............. 1.1.6......
4.1.1 Effects of the knockdown on growth charact.e.ri.s.ti.c.s .............. .............. 116
4.1.2 Effects of the knockdown on radiosensitivi.ty. .................... ................. 120
4.1.3 Effects of the knockdown on DNA damage inducdt iorne panir after
irradiation .................................................. ... 1.24..............
4.1.4 Effects of the knockdown and additional treatmithe ntte mwozolomide .. 1 26
4.2 HMEpC ........................................................ ...... 1.27.........
4.2.1 Effects of the knockdown on growth charact.e.ri.s.ti.c.s .............. .............. 127
4.2.2 Effects of the knockdown on radiosensitivi.ty. .................... ................. 128
4.2.3 Effects of the knockdown on radiation-induceda mDNaAg ed and repair . 130
4.3 Conclusions ..................................................... ..... 1.3.1.........
Appendix ............................................................. ............. 132
Reference List ........................................................ ............ 153
Poster presentations .................................................... .......... 1.7.0
Acknowledgements ..................................................... ......... 1.7.1..


Un pour Un
Permettre à tous d'accéder à la lecture
Pour chaque accès à la bibliothèque, YouScribe donne un accès à une personne dans le besoin