Cell cycle regulation in the liver: differential functions of E-type cyclins E1 and E2 for G1/S-phase transition and endoreplication in mice [Elektronische Ressource] / vorgelegt von Yulia Nevzorova

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

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Cell cycle regulation in the liver:
Differential functions of E-type Cyclins E1 and E2
for G1/S-phase transition and endoreplication
in mice


Von der Medizinischen Fakultät
der Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades einer Doktorin der
Naturwissenschaftlichen Medizin (Dr. nat. med.) genehmigte Dissertation


vorgelegt von


Yulia Nevzorova

aus Archangelsk (Russland)



Berichter: Privatdozent Dr. rer. nat. Christian Liedtke
Universitätsprofessor Dr. rer. nat. Bernhard Lüscher



Tag der mündlichen Prüfung: 19. August 2009






Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek
online verfügbar.

Eidesstattliche Versicherung


Hiermit erkläre ich, dass ich die Dissertation mit dem Titel:

„Cell cycle regulation in the liver: Differential functions of E-type Cyclins E1 and
E2 for G1/S-phase transition and endoreplication in mice“

selbstständig verfasst habe.


Die vorliegende Arbeit wurde zwischen Oktober 2005 und Januar 2009 in der
Medizinischen Klinik III des Universitätsklinikums Aachen unter der fachlichen
Betreuung von Dr. rer. nat. Christian Liedtke und der Leitung von Prof. Dr. med.
Christian Trautwein durchgeführt.
Alle benutzten Hilsmittel und zu Hilfeleistungen herangezogenen Institutionen
und Personen sind vollständig angegeben.

Ich versichere an Eides statt, dass ich diesen Promotionsantrag erstmalig
einreiche und keine früheren Versuche einer Promotion unternommen habe.

Aachen, den 15.07. 2009















Table of Contents

1. Introduction .................................................................................................. 1
1.1 The Cell cycle .................................................................... 1
1.1.1 Cyclin D as an regulatory protein ............ 2
1.1.3 Cyclin E as a „Master Cyclin“ .................. 3
1.1.4 The role of Cyclin A and B as “mitotic cyclins” ..................................................... 5
1.1.5 Living with or without cyclins and cyclin-dependent kinases .............................. 6
1.2 Liver regeneration ............................................................................................. 9
1.2.1 Initiation of liver regeneration ................................................ 11
1.2.2 The proliferation phase ........................................................... 12
1.2.3 Termination of liver regeneration .......................................... 14
1.3 Liver Fibrosis .................................................................................................. 14
1.4 Aim of the study .............................................................. 15
2. Material and methods................................................. 17
2.1 Materials .......................................................................................................... 17
2.2 Methods .................................................... 22
2.2.1 Housing and breeding of mice ............................................... 22
2.2.2 Partial hepatectomy (PH) and tissue sampling .................... 23
2.2.3. Isolation and analysis of DNA ............................................... 23
2.2.3.1. Isolation of genomic DNA from tail biopsies................ 23
2.2.3.2. Genotyping ...................................................................... 24
2.2.4 Isolation and analysis of RNA ............... 25
2.2.4.1 Isolation of RNA from liver tissue .................................. 25
2.2.4.2 Determination of RNA concentration ............................. 25
2.2.4.3 Reverse Transcription (RT-PCR) .................................... 26
2.2.5. Isolation and analysis of proteins ......... 27
2.2.5.1 Protein isolation and quantification ............................................................... 27
2.2.5.2 Western blot ...................................... 28
2.2.5.3 Immunoprecipitations and in vitro Kinase assays ...................................... 29
2.2.6 Work with primary cells .......................... 30
2.2.6.1 Isolation of hepatocytes .................................................. 30
2.2.6.2 Analysis of DNA content by Fluorescence Activated Cell Sorting (FACS) 30
2.2.6.3 Fluorescent In Situ Hybridization (FISH) ....................................................... 31
2.2.7 Quantification of liver proliferation and immunhistochemistry ......................... 31
2.2.7.1 PCNA Staining .................................................................. 31
2.2.7.2 BrdU Labeling ................................... 32
2.2.7.3 Measurement of histone H3 phosphorylation. .............. 33
2.2.7.4 Histological analysis and morphometry ........................ 33
2.2.8 Generation and in vivo use of recombinant cyclin E2 adenovirus (adv-CcnE2)
............................................................................................................................................ 34
2.2.9 Induction of Liver Fibrosis with CCl ..... 35 4
2.2.10 Sirius red staining . 35
3. Results ........................................................................................................ 36
3.1 Cyclin E1 – and cyclin E2 deficient mice have different kinetics of S-phase
initiation after partial hepatectomy ...................................... 36
-/- -/-3.2 Altered S-phase initiation in E1 - and E2 mice correlates with aberrant
expression of cyclin E1, cyclin E2 and p27 ......................................................... 41
-/- -/-3.3 Kinetics of DNA synthesis differs in E1 and E2 mice ............................... 46
3.3 Ectopic over-expression of cyclin E2 inhibits cell cycle progression in the
regenerating liver .................................................................................................. 48
-/-3.4 Increased DNA-synthesis in E2 mice results in augmented liver mass and
improved survival in a model of fatal liver failure .............................................. 53
-/-3.5 Amplified DNA synthesis in E2 mice is not correlated with increased
mitotic activity ....................................................................................................... 55
3.6 Cyclin E1 and cyclin E2 provide complementary functions for
endoreplication in hepatocytes after PH ............................................................. 57
3.7 Role of E-type cyclins in liver fibrogenesis .................................................. 62
3.7.1 Over-expression of c-myc in hepatocytes induces liver fibrosis in a cyclin E1
dependent manner ............................................................................ 62
3.7.2 Cyclin E1 is an essential pro-fibrotic factor in the liver ...... 65
3.7.3 Inhibition of Cyclin E2 accelerates the onset of liver fibrogenesis .................... 68
4. Discussion .................................................................. 70
4.1 Role of E-type cyclin during liver regeneration ............................................ 70
4.2 Role of E-type cyclins during fibrogenesis ................... 74
5. References .................................................................. 78
6. Summary ..................................... 86
7. Appendix ..... 88
7.1 Abbrevitions .................................................................................................... 88
7.2 Publications .............................................. 90
7.3 Acknowledgements ........................................................................................ 92
7.4 Erklärung zur Datenaufbewahrung ................................................................ 92
7.5 Curriculum Vitae ............................................................................................. 92






1. Introduction 1

1. Introduction
1.1 The cell cycle
The cell cycle is an ordered set of events eventually culminating in cell
growth and division into two daughter cells. In virtually in all cells, the cell cycle
is composed of four discrete phases, which are the DNA synthesis phase (S
phase), the mitotic phase (M phase) and the intermediate gap phases referred
to as G1-phase (between M and S phases) and G2-phase (between S and M
phases).
During the last decades, a wealth of knowledge has become available
giving insight into the molecular mechanisms controlling cell cycle regulation.
Cyclins and their partners, the cyclin dependent kinases (CDKs) constitute
the basis of these molecular mechanisms (Boonstra, 2003).

quiescent cells Fig. 1 Overview of the cell
cycle in mammalian cells. (G0)

As cells proceed through the
cycle, four major cyclins are
produced sequentially (D, E, A,
Cyclin D and B), and activate different
Cyclin B CDKs. After progression CDK4,6, through the restriction point G1CDK1 (R) cells ente