Isoform-specific loss of CD44 interferes with different aspects of the metastatic process [Elektronische Ressource] / presented by Pamela Klingbeil

Sujets

• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the
Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences










presented by
Dipl. Biol. Pamela Klingbeil
born in Berlin


Oral-examination:









Isoform-specific Loss of CD44 Interferes with
Different Aspects of the Metastatic Process


















Referees: PD Dr. Jochen Wittbrodt
Prof. Dr. Margot Zöller




























Whenever you fall,
pick something up.

Oswald Theodore Avery Table of Contents
Table of Contents

Summary.................................................................................................................................. 1
Zusammenfassung.................................................................................................................... 2
1. Introduction.................................................................................................................. 4
1.1 Cancer evolves as a multistep process.................................................................................. 4
1.2 The BSp73 cell system......................................................................................................... 12
1.3 The cell-cell and cell-matrix adhesion molecule CD44....................................................... 13
1.3.1 Structural properties of CD44......................................................................................... 13
1.3.2 Different modes of interactions for CD44...................................................................... 16
1.3.3 Physiological and pathological functions ascribed to CD44.......................................... 18
1.3.4 CD44 in tumor progression............................................................................................. 20
1.4 RNAinterference as a tool to study isoform specific gene functions................................... 22
1.5 Aims of the thesis................................................................................................................. 23
2. Results........................................................................................................................... 25
2.1 Establishment of stable CD44vk.d. cell lines and rescue clones......................................... 26
2.1.1 RNAi construct evaluation by FACS and fluorescence microscopy.............................. 26
2.1.2 Establishment of stable CD44vk.d. clones by selection and recloning.......................... 27
2.1.3 Restoring CD44 expression by introduction of mutated cDNAs................................... 28
2.2 Characterization of the knock-down cell lines in vivo......................................................... 29
2.2.1 CD44vk.d. cells exhibit a reduced metastatic capacity in vivo....................................... 29
2.3 Characterization of knock-down cell lines in vitro.............................................................. 33
2.3.1 CD44vk.d. cells show no phenotypic changes................................................................ 33
2.3.2 CD44vk.d. cells show no altered growth behaviour....................................................... 33
2.3.3 CD44vk.d. cells do not differ in MMP2 and MMP9 expression.................................... 34
2.3.4 ASMLwt but not CD44vk.d. cells aggregate in stromal cell culture supernatant.......... 35
2.3.5 ASMLwt cells produce an adhesive matrix, which is impaired in the CD44vk.d......... 37
2.3.5.1 Adhesion promoting components are secreted.......................................................... 39
2.3.5.2 The secreted matrix contains HA, collagen and laminin........................................... 40
2.3.5.3 Adhesion to the secreted matrix is mediated by β1 integrin...................................... 43
2.3.6 CD44vk.d. cells lack a secreted 180 kDa protein........................................................... 44
2.3.7 CD44vk.d. cells exhibit a reduced resistance to apoptotic triggers................................ 45
2.3.7.1 Apoptosis resistance is increased by elongated pre-cultivation prior to irradiation.. 46
2.3.7.2 PI3K-Akt, rather than MAPK signalling is involved in apoptosis resistance of
ASML cells................................................................................................................ 48
3. Discussion..................................................................................................................... 52
4. Materials and Methods............................................................................................... 66
4.1 Materials............................................................................................................................... 66
4.1.1 Chemicals........................................................................................................................ 66
4.1.2 Enzymes.......................................................................................................................... 67
4.1.3 Chemical inhibitors......................................................................................................... 67
4.1.4 Nucleotide and protein standards.................................................................................... 67
4.1.5 Kits.................................................................................................................................. 67
4.1.6 Vectors............................................................................................................................ 68
4.1.7 Primers and oligos........................................................................................................... 68
4.1.8 cDNAs and constructs..................................................................................................... 69
4.1.9 Antibodies....................................................................................................................... 69
4.1.9.1 Primary antibodies..................................................................................................... 69
4.1.9.2 Secondary antibodies/ reagents.................................................................................. 70 Table of Contents
4.1.10 Cell lines ......................................................................................................................... 71
4.1.11 Animals........................................................................................................................... 71
4.2 Methods................................................................................................................................... 71
4.2.1 Molecular biology........................................................................................................... 71
4.2.1.1 Bacteria...................................................................................................................... 71
4.2.1.2 Plasmid preparation................................................................................................... 72
4.2.1.3 RNAinterference construct design and cloning......................................................... 72
4.2.1.4 PCR-based mutagenesis for rescue constructs .......................................................... 73
4.1.2.5 RNA-isolation and reverse transcription-PCR (RT-PCR)......................................... 73
4.2.2 Cell biology..................................................................................................................... 74
4.2.2.1 Cell culture................................................................................................................. 74
4.2.2.2 Cryo-conservation of eukaryotic cells....................................................................... 74
4.2.2.3 Transfection of eukaryotic cells................................................................................ . 74
4.2.2.4 Recloning of transfected cells by limiting dilution.................................................... 74
4.2.2.5 Collection of conditioned cell culture supernatant.................................................... 75
4.2.2.6 Coating of plastic surfaces......................................................................................... 75
4.2.2.7 Adhesion assay........................................................................................................... 75
4.2.2.8 Agglomeration assay.................................................................................................. 76
4.2.2.9 Proliferation assay.........................................................................