Molecular characterization of TGF-β [TGF-beta] mediated cancer cell proliferation [Elektronische Ressource] / vorgelegt von Garima Singh

Shiv_2

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Aus dem Medizinischen Zentrum für Innere cMhewdeizrinp,u nSkt Gastroenterologie
und Stoffwechsel
Direktor: Prof. Dr. med. Thomas Gress
des Fachbereichs Medizin in Zusammenarbeit mUitn ivdermsi tätsklinikum Gießen und
Marburg GmbH, Standort Marburg

Molecular characterization of TGF-β
mediated cancer cell proliferation








Inaugural-Dissertation zur Erlangung des Doktoergsr adder gesamten Humanmedizin
dem Fachbereich Medizin der Philipps-Universiträbtu rMga
vorgelegt vo n


Garima Singh
aus Muzaffarnagar, India

Marburg, 2010
I




















Angenommen vom Fachbereich Medizin der Philipvpes-rUsintäit
Marburg am
15 April 2010
Gedruckt mit Genehmigung des Fachbereichs
Dekan: Prof. Dr. med. Matthias Rothmund
Referent: Prof. Dr. Volker Ellenrieder
Korreferent: Prof. Dr. Thorsten Stiewe


II










I dedicate my thesis to my uncle late
Dr. U. V. Singh, my husband Shiv Kishor Singh
and my family






III

Table of Contents
SUMMARY....................................................... ................... VI
ZUSAMMENFASSUNG................................................. ...........V.I.I.I...
1 INTRODUCTION....................................................................................... ..................................1
1.1 THE PLURIPOTENCY OF TRANSFORMING GROWTH FACTOR - BETA.................................. .....1............
1.2 TGF-ΒETA SIGNALLING ACTIVATION ...................................................1. ........................
1.3 THE SMAD SIGNALLING PATHWAY IN N ORMAL AND M ALIGNANT EPITHELIAL CELLS..................... .................2
1.4 GENETIC ALTERATIONS OF THE SMAD SIGNALLING PATHWAY .................................... ........4.......
1.5 SMAD -INDEPENDENT SIGNALLING IN N ORMAL AND M ALIGNANT EPITHELIAL CELLS...................... ...................6
1.6 TGF-ΒETA GROWTH CONTROL IN NORMAL AND MALIGNANT CELLS ................................. ....8..............
1.7 M ECHANISMS UNDERLYING THE DUAL ROLE OF TGF-ΒETA DURING TUMORIGENESIS ...................... ................10
2 AIMS OF THE STUDY ................................................ .................1.3
3. MATERIALS AND METHODS...................................................................... ........................1.4......
3.1 M ATERIALS...................................................................... ............14
3.1.1 Cell Lines......................................................................... ..............14
3.1.2 Chemical reagents and general materials.......................................... .....1.4...
3.1.3 Chemical reagents................................................................................................... ............15
3.1.4 Transfection reagents............................................................. ..........17
3.1.5 Radioactive chemical............................................................. ..........17
3.1.6 Instruments....................................................................... ............18
3.1.7 Kits.............................................................................. ...............19
3.1.8 PCR reagents........................................................................................................... ............19
3.1.9 Real-time PCR primers for cDNA................................................ .......19
3.1.10 Real-time primers for ChIP...................................................... ........20
3.1.11 Expression vectors............................................................... ...........21
3.1.12 List of antibodies for Western blot ana.l.y.s.is....................................... ......2.2.
3.1.13 List of antibodies for ChIP analysis....................................................................... .............22
3.2 SOLUTIONS AND BUFFERS ............................................................ ....2.3............
3.2.1 Mediums and buffer solutions.................................................... .......23
3.2.1.1 Cell biological................................................................ 2.3.....................
3.2.1.2 Biochemical..................................................................2. 4......................
3.2.1.3 Reagents for ChIP analysis.......................................................................................... .......................27
3.3 M ETHODS ...................................................................... .............29
3.3.1 Preparation of competent cells ( CmaCelthod)........................................ ......292
IV

3.3.2 Transformation of competent bacteria.................................................................. ...........2.9.
3.3.3 Bacterial manipulation............................................................ ..........30
3.4 EXPERIMENTAL PROCEDURES .......................................................... ..3.1................
3.4.1 Cells and transfection............................................................ ...........31
3.2.4 Preparation of whole protein extract from mmaaliman cells.................................. 3..1.........
3.2.5 Preparation of nuclear and cytoplasmic pnr oetxetiracts from mammalian cells......... .....32
3.2.6 Protein determination............................................................ ..........33
3.2.7 SDS-polyacrylamide gel electrophoresis........................................... ......3.3.
3.2.8 Western blotting................................................................. ...........33
3.2.9 siRNA transfection.................................................................................................... ...........34
3.2.10 Proliferation assay and cell cycle anal.y.s.is......................................... .......34
3.2.11 RT-PCR......................................................................... .............35
3.2.12 DNA pull-down................................................................ ...........35
3.2.13 Chromatin Immunoprecipitation Analysis ()C.h.IP.................................... ...3.6........
3.2.14 Luciferase Reporter Assays................................................................................... ............37
3.2.15 Statistical analysis.............................................................. ............37
4 RESULTS......................................................... ....................43
4.1 TGF-ΒETA PROMOTES CELL PROLIFERATION VIA G1/S PHASE PROGRESSION IN CANCER CELLS ............... ..........43
4.2 C-M YC IS REQUIRED FOTRG F-ΒETA INDUCED CANCER CELL PROLIFERATION ........................... ......................46
4.3 NFAT TRANSCRIPTION FACTORS ARE ESSENTIAL FOR C-M YC INDUCTION BY TGF-ΒETA IN CANCER CELLS ......... ...49
4.4 TGF-ΒETA MODULATES NFAT ACTIVITY IN SAM AD -INDEPENDENT MANNER ........................... .....................55
4.5 CALCINEURIN DEPENDENT TGF-ΒETA MEDIATED NFAT INDUCTION ................................ ..5..8...............
4.6 REQUIREMENT OF NFAT FOR TGF-ΒETA MEDIATED -CM YC INDUCTION IN CANCER ...................... ................61
4.7 NFAT ANTAGONIZES THE SMAD 3 REPRESSOR COMPLEX TO ACTIVATE THE C -M YC PROMOTER ............... ..........65
4.8 NFAT MEDIATES THE TGF-ΒETA SWITCH FROM A GROWTH SUPPRESSOR TO A PROMOTER OF CELL PROLIFERATION
.................................................................................... ..6..8...............
5 DISCUSSION .................................................... ...................71
5.1 SIGNIFICANCE OF MY WORK :.......................................................................................... ....7.9...............
6. REFERENCES................................................... ...................81
7 ABBREVIATIONS.................................................. ..................92
8 ACKNOWLEDGEMENTS............................................................................. .........................9.5.....
9 CURRICULUM VITAE ............................................... ................9.8.
V
Summary
SUMMARY


TGF-β inhibits epithelial cell growth through dSempaed-ndent induction of a cell cycle
arrest at G1. During tumor progression, howenvye tru,m mora cells escape from TGF-β
growth suppression due to either functionalt ico rd igserunpetion of the Smad signaling
pathway. In late tumor stages, TGF-β stimulatoer s cetullm proliferation through
increased cell cycle transition. Although thuisl arc eellvent is clearly established, the
molecular mechanismus nderlying this phenomenon remain unknown. He srheo,w we
that TGF-β stimulation induces cancer cell praotliiofn evria accelerated G1/S phase
transition. We show that cell proliferation sr einqduuircetion of the c-Myc oncogene,
and this is paralleled by upregulation of Dcli-ntsy paen d cytheir corresponding CdKs.
TGF-β induces c-Myc expression on the level ootfer prreomgulation through induction
of the c-Myc/TIE element, which has previo ursely pobretedn as the core element for
repression of c-Myc in growth inhibited