Cell culture models and novel gene therapeutic strategies for colorectal cancer [Elektronische Ressource] / vorgelegt von Lars Gädtke

Lars Gaedtke

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Dissertation
zur Erlangung des Doktorgrades der Fakultät für Chemie und
Pharmazie der Ludwig-Maximilian-Universität München



Cell culture models and novel gene therapeutic strategies for
colorectal cancer

vorgelegt von
Lars Gädtke
aus Leonberg
2006

Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Prof. Dr. Ernst Wagner betreut.



Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München,



_ _ _ _ _ _ _ _ _ _ _ _ _ _
(Lars Gädtke)



Dissertation eingereicht am 12.01.2006
1. Gutacher: Prof. Dr. Ernst Wagner
2. Gutacher: PD. Dr. Carsten Culmsee
Mündliche Prüfung am 15.02.2006

Table of Contents
1. INTRODUCTION..................................................................................................8
1.1. Colorectal cancer ..........................................................................................8
1.1.1. Pathogenesis of colorectal cancer ..............................................................8
1.1.2. Human low passage colon cancer cell lines..............................................10
1.1.3. Multicellular tumor spheroids.....................................................................11
1.2. Chemotherapy of colorectal cancer ..........................................................13
1.2.1. Chemoresistance against chemotherapeutic drugs ..................................13
1.2.2. Mechanisms of action of 5-fluorouracil......................................................14
1.3. Gene therapy of colorectal cancer.............................................................16
1.3.1. Gene delivery strategies ...........................................................................16
1.3.2. Tumor specific cell targeting strategies .....................................................17
1.3.3. Therapeutic genes ....................................................................................18
1.4. Specific aims of the PhD thesis .................................................................20
1.4.1. Initiation and characterization of relevant model systems for
colorectal cancers .....................................................................................20
1.4.2. Elucidation of mechanisms involved in resistance of colorectal
cancers against chemotherapy20
1.4.3. Development of alternative therapy strategies for colorectal cancers .......21
2. MATERIAL AND METHODS .............................................................................22
2.1. Chemicals and reagents22
2.2. Molecular biological methods....................................................................23
2.2.1. Restriction digestion of plasmid DNA ........................................................23
2.2.2. Dephosphorylation of plasmid DNA fragments..........................................23
2.2.3. Converting of 5´- overhangs of DNA fragments to blunt ends...................23
2.2.4. Isolation of DNA fragments from agarose gels..........................................23
2.2.5. Ligation .....................................................................................................23
2.2.6. Transformation of E.coli ............................................................................24
2.2.7. Preparation of plasmid DNA......................................................................24
2.2.8. Cloning strategies .....................................................................................24


2.3. Cell biological methods ..............................................................................25
2.3.1. Cell culture ................................................................................................25
2.3.2. Multicellular spheroid culture.....................................................................26
2.3.3. Formation of transfection complexes ........................................................26
2.3.4. Measurement of particle size and zeta-potential.......................................27
2.3.5. Gene transfer to monolayer cultures .........................................................27
2.3.6. fer to multicellular spheroids....................................................28
2.3.7. Luciferase assay .......................................................................................28
2.3.8. Human IL-2 ELISA ....................................................................................29
2.3.9. Treatment of cells with 5-fluorouracil29
2.3.10. Proliferation and viability assays ...............................................................29
2.3.10.1. Hoechst 33258-based proliferation assay.............................................29
2.3.10.2. MTT assay ............................................................................................30
2.3.11. Flow cytometric analysis ...........................................................................30
2.3.11.1. Flow cytometric analysis of EGFP expression ......................................30
2.3.11.2. Flow cytometric analysis of apoptosis...................................................30
2.3.12. Transmission light and epifluorescence microscopy .................................31
2.3.13. Confocal laser scanning microscopy of multicellular spheroids.................32
2.3.14. Cryosections of multicellular spheroids .....................................................32
2.4. 2D Electrophoresis and MALDI-TOF mass spectrometry........................33
2.4.1. Sample preparation...................................................................................33
2.4.2. Measurement of protein concentration......................................................33
2.4.3. First dimension: Isoelectric focusing .........................................................33
2.4.4. Second dimension: SDS-page ..................................................................34
2.4.5. Silver staining............................................................................................35
2.4.6. 2D image analysis.....................................................................................35
2.4.7. In-gel digestion..........................................................................................36
2.4.8. Desalting and spotting of peptides onto the MALDI-TOF MS target..........36
2.4.9. MALDI-TOF mass spectrometry and peptide mass fingerprinting.............37
3. RESULTS ..........................................................................................................38
3.1. Multicellular spheroids of low passage colon cancer cell lines -
A promising model system for colorectal cancer ....................................38
3.1.1. Establishment of multicellular spheroids of low passage colon cancer
cell lines ....................................................................................................38
3.1.2. Differences in the expression profiles of multicellular spheroids
compared to corresponding monolayer cultures .......................................39
3.2. Chemotherapy of colorectal cancer – Detection of proteins
associated with chemoresistance against 5-FU50
3.2.1. Determination of 5-FU concentrations required for reduction of
proliferation of selected low passage colon cancer cells...........................50
3.2.2. Long-term 5-FU treatment of selected low passage colon cancer cells ....53
3.2.3. Effect of 5-FU on proliferation of the long-term 5-FU-pretreated sublines.54
3.2.4. Effect of 5-FU on the induction of apoptosis in the long-term 5-FU
pretreated subline COGA-12/G6...............................................................55
3.2.5. Effect of 5-FU on proliferation and apoptosis in long-term propagated
COGA-12 cells ..........................................................................................56
3.2.6. Differences in the expression profiles of chemoresistant cells
compared to corresponding chemosensitive cells.....................................58
3.3. Gene therapy of colorectal cancer.............................................................67
3.3.1. Optimization of nonviral gene transfer to colorectal cancer cells...............67
3.3.1.1. Generation and biophysical properties of nonviral gene transfer
formulations67
3.3.1.2. Determination of the efficiencies of the most adequate
formulations in gene transfer.................................................................69
3.3.1.3. Transfection of multicellular spheroids with lipopolyplexes ...................71
3.3.2. Transcriptional targeting of colorectal cancer cells....................................75
3.3.2.1. Gene expression levels after transcriptional targeting in various
low passage colon cancer cell lines......................................................75
3.3.2.2. Transfection of selected low passage colon cancer cell lines with
transcriptionally targeted lipopolyplexes ...............................................77
3.3.2.3. Influence of plasmid DNA concentration on gene expressi