MTBITC-induced apoptosis and cell cycle arrest of human hepatoma (HepG2) cells [Elektronische Ressource] : a link between p53 and human telomerase ? / vorgelegt von Evelyn Lamy

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2008
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 2 Mo

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MTBITC-induced apoptosis and cellMTBITC-induced apoptosis and cell
cycle arrest of human hepatoma
(HepG2) cells:(HepG2) cells:
A link between p53 and human telomerase ?
Inaugural - Dissertation
zur Erlangung des Grades einer Doktors der Humanbiologie (Dr. biol. hom.)
des Fachbereichs Humanmedizin
der Justus-Liebig-Universität Gießen
vorgelegt von: Evelyn Lamy
aus: Saarbrücken
Giessen im Jahr 2007Aus dem Institut für Innenraum- und Umwelttoxikologie
des Fachbereichs Humanmedizin der Justus-Liebig-Universität Gießen
Direktor: Prof. Dr. V. Mersch-Sundermann
1. Gutachter: Univ. Prof. Dr. med. V. Mersch-Sundermann
2. Gutachter: Univ. Prof. Dr. med. A. Reiter
Tag der Disputation: 26.5.2008 Meinen Eltern & Alexander
Es ist nicht genug zu wissen, man muss es auch anwenden; es ist nicht genug zu wollen,
man muss es auch tun.
Johann Wolfgang von GoetheAcknowledgements i
ACKNOWLEDGEMENTS
This thesis grew out of a series of dialogues with my supervisor Professor Dr. Volker
Mersch-Sundermann, who gave me every opportunity to broaden my educational horizon
and to develop my personality. He piqued my curiosity and provoked me in his very
special manner to keep on track. I honestly could not have imagined having had a better
mentor; his common-sense, his occasional cracking-of-the-whip, but also his patience with
me is greatly appreciated. Especially his axioms will inspire me - or rather haunt me - for
the rest of my life.
I thank all my research colleagues for providing a stimulating and fun environment in
which to learn and grow. In particular, I wish to thank Dr. Richard Gminski, Ariane
Ollman, Yvonne Völkel, Julia Schröder and Peter Brenk. Furthermore, I am indebted to
Tao Tang, who made my life easier during the sometimes hard days in the lab. I will truly
miss his all-inclusive catering. I especially would like to thank Dr. Thorsten Stahl (LHL,
Wiesbaden) for wonderful conversations and the weekly dose of vitamin C.
Dr. Xinjiang Wu (University of Philadelphia, Faculty of Cancer Biology, USA) gave me a
helping hand with the immunoblot technique and flow cytometry and Dr. Simone Helmig
and Juliane Döhrel (Institute of Occupational Health Medicine, JLU Giessen) helped me
with some molecular biology issues. They all gave me important thought-provoking
impulses. Thanks to Prof. Dr. Peter Schreiner and Dr. Mike Kotke (Institute of Organic
Chemistry, JLU Giessen) for the synthesis of MTBITC and to Prof. Dr. Firouz Darroudi
(LUMC, the Netherlands) for providing the HepG2 cells. I also want to thank Ms. Deborah
Lawrie-Blum (IUK, Freiburg) for proofreading this thesis.
Finally, I am forever indebted to my parents Horst and Monika Lamy and Alexander for
their understanding, endless patience and constant encouragement and love I have relied
upon throughout this time. I am also grateful to Mathilde, who is sitting right here beside
me again, wondering curiously with her beautiful red eyes at what I might be doing.
On a different note, I would like to thank the black tea producers of India for keeping me
awake, and the fair trade organization for making me feel okay about drinking so much tea.
Last but not least, I am really thankful to my bicycle for keeping me fit and my heating for
keeping me warm at the desk during the cold winter days in the office, although my male
colleagues will probably shake their heads in complete bewilderment when reading this. Table of Contents ii
ACKNOWLEDGEMENTS ..................................................................................................i
TABLE of CONTENTS.......ii
LIST of ABBREVIATIONS ................................................................................................v
I. ABSTRACT ....................................................................................................................1
II. INTRODUCTION...........4
2.1 ITCs in Disease and Cancer Prevention ....................................................4
2.2 4-Methylthiobutylisothiocyanate (MTBITC).............4
2.3 Chemopreventive properties of ITCs.........................5
2.4 Apoptosis induction and cell cycle regulation by ITCs.............................5
2.5 The p53 family ..............................................................................................6
2.6 The relationship between telomeres, telomerase and p53.........................6
2.7 Objectives and hypothesis............................................................................8
III. MATERIAL .................................................................................................................9
3.1 The human cell culture model HepG2........................9
3.1.1 Biochemical characterization .........................................................................9
3.1.2 Tumor suppressor p53 status........10
3.2 General material.........................................................................................10
3.3 Chemicals....................................11
3.4 Cell culture reagents...................12
3.5 Antibodies....................................................................12
3.6 Electrophoretic reference bands...............................13
3.7 Assay kits.....................................13
3.8 Staining solutions........................................................................................14
3.9 Equipment and software............14
3.10 Media, buffers and solutions .....................................................................15
3.11 Test substance and controls.......18Table of Contents iii
IV. METHODS.................................................................................................................21
4.1. General cell culture....................21
4.1.1 Cryoconservation & reanimation of the cell culture ....................................21
4.1.2 Cell culture and passage ...............................................21
4.1.3 Procedure of chemical exposure...21
4.2 Assessment of cell proliferation and viability .........................................22
4.3 Techniques for the assessment of apoptosis induction............................23
4.3.1 DNA Laddering ...........................................................................................23
4.3.2 Measurement of the “subG1 DNA” content.................24
4.3.3 Measurement of single stranded apoptotic DNA (ssDNA assay) ................25
4.4 Measurement of the cell cycle distribution...............................................26
4.5 Detection of DNA damage by the Comet assay........27
4.6 Analysis of the mitochondrial membrane potential (MMP)...................28
4.7 Determination of reactive oxygen species (ROS) by DHR123................29
4.8 Determination of the glutathione status ...................................................31
4.9 Telomerase activity measurement by TRAP-ELISA ..............................32
4.10 Telomere length assessment by flow-FISH..............35
4.11 Protein analysis by immunoblotting .........................................................38
4.12 Determination of the protein concentration after Bradford ..................40
4.13 Chemical analysis by GC-MS/MS.............................41
4.14 Statistical data analysis ..............................................................................42
V. RESULTS...................................................................................................................43
5.1 MTBITC inhibits the proliferation of HepG2 cells .................................43
5.2 Apoptosis is induced in a concentration- and time-dependent manner 45
5.3 The cell cycle progression is halted by MTBITC.....48
WAF15.4 p21 is increased by MTBITC-treatment...........................................49
5.5 MTBITC directly modifies the MMP.......................50
5.6 The anti-apoptotic protein BCL is suppressed......52XL
5.7 The GSH level of HepG2 cells is dichotomous modulated......................53
5.8 ROS production is a late event in MTBITC-induced apoptosis.............55Table of Contents iv
5.9 The p53 family is involved in MTBITC-mediated growth suppression 56
5.10 Overexpression of murine double minute (MDM2) oncogene ...............59
5.11 MTBITC induces DNA migration in the Comet assay ...........................60
5.12 Telomerase and telomerase activity are suppressed by MTBITC.........61
5.13 MTBITC-treatment increases the level of HSP proteins........................63
5.14 The telomere length of HepG2 cells is not rapidly decreased.................65
5.15 MTBITC degrades rapidly in the experimental system .........................66
5.17 Only early removal of MTBITC saves the cells from apoptosis.............69
VI. DISCUSSION..........................................................................................................71
6.1 DNA damage is an initial event in MTBITC-mediated growth
inhibition ..................................................................................................