Notch 1 -dependent regulation of cell fate in colorectal cancer

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1 Notch 1 -dependent regulation of cell fate in colorectal cancer Referees: PD Dr. Tobias Dick Prof. Dr. Wilfried Roth

2 CONTENTS Summary 1 Zusammenfassung 2 1 INTRODUCTION Colorectal cancer Pathogenesis Classification Epidemiology and risk factors Diagnosis and therapy Cell cycle Cyclin-dependent kinase inhibitors p27 Kip p21 Cip Cellular senescence Apoptosis Caspases Extrinsic apoptotic pathway Intrinsic apoptotic pathway Apoptosis and cancer Notch signaling Mechanism of Notch signaling 19 I

3 1.4.2 Notch signaling and cancer 21 Aims of the work 23 2 MATERIALS Cell lines Chemicals Common buffers, solutions and media Buffers for immunoblot analysis Buffer for agarose gel electrophoresis Staining solutions Loading buffers FACS buffer Media for culturing and freezing Escherichia coli Buffers for KCM-competent E. coli Top Antibodies Oligonucleotides sirna PCR primers qrt-pcr primers Sequencing primers Plasmids Equipment 30 3 METHODS 31 II

4 3.1 Cell biology methods Culturing human CRC cell lines Storage of CRC cell lines Transfection of sirna and plasmid DNA Adenoviral transduction Clonogenicity assay Cell counting using trypan blue exclusion Microbiological methods Generation of KCM-competent E. coli Top10 bacteria Transformation of KCM-competent E. coli Top10 bacteria Transformation of MAX efficiency DH5a competent bacteria Molecular biology methods Enzymatic restriction Ligation PCR with Phusion polymerase DNA sequencing Isolation and reverse transcription of RNA Quantitative real-time PCR Analyzing qrt-pcr primer efficiency Biochemical methods PCR primer design Agarose gels Gel extraction 37 III

5 3.4.4 Preparation of lysates Total lysates Subcellular fractionation Immunoblot analysis FACS analysis Determining cell death using Pl-staining Cell cycle analysis Brdll assay Senescence assay Immunohistochemistry Pulse-chase analysis 41 4 RESULTS Characterization of Notchl-dependent regulation of p Analysis of NICD1 and p27 levels in CRC cell lines Notchl knockdown causes upregulation of p Notchl regulates p27 levels post-transcriptionally Notchl controls p27 half-life Notchl modulates p27 proteasomal degradation by controlling the expression of p27-targeting E3 ubiquitin-protein ligase subunits SKP2, KPC1 and KPC Knockdown of Notchl induces a decrease of the protein levels of SKP2, KPC1 and KPC The regulation of SKP2, KPC1 and KPC2 by Notchl occurs at least partially on transcriptional level 46 IV

6 4.2.3 The regulation of SKP2 and p27 by Notch 1 signaling is additionally demonstrated by shrna-mediated Notchl depletion The knockdown of Notchl leads to increase of p27 levels in the cytoplasm as well as the nucleus The knockdown of SKP2 and KPC1 causes similar upregulation of p27 as Notchl depletion Transient overexpression of SKP2 but not KPC2 decreases p27 levels Notchl-dependent regulation of p27 is probably not mediated by p Role of p27 in Notchl-dependent cell fate decisions p27 knockdown partially rescues the decrease in cellular proliferation mediated by Notchl downregulation p27 knockdown partially rescues the decrease in colony formation mediated by Notchl downregulation p27 knockdown partially rescues the G2/M phase arrest mediated by Notchl downregulation Regulation of p21 by Notchl signaling might contribute to the p27- mediated effect of Notchl on cell fate Overexpression of p27 does not resemble Notchl effect on cell cycle Clinical relevance of p27 regulation by Notchl Low expression of p27 is associated with high proliferative capacity Low p27 expression and high Notchl expression are observed at the infiltration zones of colorectal carcinomas Therapeutic significance of Notchl inhibition Notchl-depleted cells undergo cell senescence Notchl knockdown alone causes CRC cell death 58 V

7 4.4.3 Notch 1 knockdown enhances the effect of chemotherapy Notchl knockdown enhances the effect of radiotherapy Notchl knockdown enhances the effect of small-molecule inhibitors used in the clinical practice HCT116 cells respond differently to Notchl knockdown Notchl regulation of apoptosis Kinetics of activation of apoptosis by Notchl knockdown shrna- and sirna-mediated Notchl knockdown causes downregulation of the anti-apoptotic proteins Bcl-X L and Mcl DISCUSSION Regulation of cell cycle by Notchl signaling Regulation of cellular senescence by Notchl signaling Inhibition of apoptosis by Notchl signaling Therapeutic significance of Notchl signaling inhibition 76 6 REFERENCES 81 Abbreviations 101 Acknowledgements 107 List of publications 108

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