Analysis of enhanced retinoblastoma protein (pRb) degradation in HPV-positive cells after histone deacetylase inhibition [Elektronische Ressource] / presented by Handan Karaduman

Karaduma

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Analysis of enhanced retinoblastoma protein (pRb)
degradation in HPV-positive cells after histone
deacetylase inhibition







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




presented by

Handan Karaduman

from Trabzon, Turkey



June 2008





DISSERTATION

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













presented by


Diplom-Biologin Handan Karaduman

born in: Karlsruhe, Germany











Oral examination: ......................................................






Analysis of enhanced retinoblastoma protein (pRb)
degradation in HPV-positive cells after histone deacetylase
inhibition
















Referees: PD. Dr. Dieter Kübler

Prof. Dr. Frank Rösl



















THE MILL, THE STONE, AND THE WATER

All our desire is a grain of wheat.
Our whole personality is the milling-building.
But this mill grinds without knowing about it.

The millstone is your heavy body.
What makes the stone turn is your thought-river.
The stone says: I don’t know why we do all this,
but the river has knowledge!

If you ask the river, it says,
I don’t know why I flow,
All I know is that a human opended the gate!

And if you ask the person, he says:
All I know, oh gobble of bread, is that if this
stone stops going around, there’ll be no bread for your bread-soup!
All this grinding goes on, and no one has any
knowledge!
So just be quiet, and one day turn to God, and say:
” What is this about bread-making?”


Hz. Mevlana Celaleddin Rumi

1
INDEX

Index ..................................................................................................................................... 1
Abbreviations ……………………………………………………………………………... 5
Abstract ………………………………………………………………………………......... 7
Zusammenfassung ………………………………………………………………………… 9

I. INTRODUCTION ……………………………………………………………………... 11
1.1 Human papillomavirus (HPV) onfection can cause benign and malignant tumors on
squamous epithelial cells …………………………………………………………………... 11
1.2 Cervical cancer is causally linked with HPV infection ………………………………… 11
1.3 Functional organization of the HPV Genome ………………………………………….. 12
1.4 HPV oncoprotein and their transformation capacity …………………………………… 13
1.4.1 The high-risk E6 oncoprotein and its biological activities …………………… 14
1.4.2 The high-risk HPV E7 oncoprotein and its biological activities ……………... 15
1.4.2.1 Control of the cell cycle and its disruption by high-risk HPV E7 ….. 16
1.4.2.2 Binding of High-risk HPV E7 to tumor suppressor protein pRb
abrogates the control for transition from G1 to S Phase during the cell cycle ... 18
1.5 The tumorsuppressor protein “Retinoblastoma Protein” ………………………………. 20
1.5.1 The tumor suppressor protein pRb and Cancer ……………………………….. 20
1.5.2 pRb belongs to the pocket protein family and exerts different function ………. 20
1.5.3 Regulation of the E2F transcription factors by pRb ………………………….. 22
1.5.4 Transcriptional repression by the pRb/HDAC/E2F complex ………………… 23
1.6 Chromatin regulation and the role of HDAC inhibitors ………………………... 25
1.6.1 Chromatin regulation by HATs and HDACs ………………………………… 25
1.6.1 Inhibitors of HDAC molecules ……………………………………………….. 26
1.6.3 HDAC inhibitors induced cellular effects ……………………………………. 26
1.6.3.1 Influence of HDAC inhibition on gene transcription ………………. 27
1.6.3.2 HDAC and cell cycle …………………………………………......... 28
1.6.3.3 HDAC inhibitor induced apoptosis ………………………………… 28
1.6.4 HDAC inhibitors and treatment of cancer …………………………………… 29

Aims of this study ................................................................................................................ 32


2

II. MATERIALS AND METHODS ……………………………………………..……….. 33
2.1 Materials ……………………………………………………………………………….. 33
2.1.1 Chemicals and Reagents ……………………………………………………… 33
2.1.2 Laboratory equipment ………………………………………………………... 35
2.1.3 Solutions and Buffers …………………………………………………..……… 36
2.1.4 Cell Lines ……………………………………………………………….…….. 40
2.1.5 Antibodies …………………………………………………………………….. 42
2.1.6 PCR primers …………………………………………………………………... 43
2.1.7 Plasmids ……………………………………………………………………… 43
2.1.8 Oligonucleotids for Northern Blot analyses ………………………………….. 44
2.1.9 Size markers for DNA/RNA electophoresis
2.1.10 Kits …………………………………………………………………………… 44
2.1.11 Others ………………………………………………………………………... 45
2.2 Methods ………………………………………………………………………………… 46
2.2.1 Cell culture techniques ………………………………………………………... 46
2.2.1.1 Cell culture ………………………………………………………….. 46
2.2.1.2 Thawing and freezing of eukaryotic cells …………………………... 46
2.2.1.3 Cell Counting ………………………………………………………. 46
2.2.1.4 Cell treatment with sodium butyrate, MG-132, TLCK ……………... 47
2.2.2 Preparation of DNA probes …………………………………………………… 47
2.2.2.1 Transformation of Component bacteria ……………………………... 47
2.2.2.2 Plasmid –DNA restriction analyses ………………………………… 47
2.2.2.3 DNA extraction from agarose gel (QIAquick gel extraction kit) …… 47
2.2.3 Protein analysis ………………………………………………………..……… 48
2.2.3.1 Preparation of nuclear and cytoplasmic proteins …………………….......... 48
2.2.3.2 SDS-total protein extracts ………………………………….……….. 49
2.2.3.3 SDS-Polyacrylamide gel electrophoresis ………………….………... 49
2.2.3.4 Western Blot “Semidry” …………………………………….……… 49
2.2.3 Nucleic acid analysis ………………………………………………….………. 50
TM 2.2.4.1 RNA extraction (Absolutely RNA RT-PCR Miniprep Kit) .……... 50
2.2.4.2 DNA extraction from eukaryotic cells …………………………..….. 50
2.2.4.3 DNA restriction analysis ……………………………………………. 51
2.2.4.4 DNA electrophoresis and Southern Blot …………………….……… 51
2.2.4.4 RNA electrophoresis and Northern Blot ……………………………. 52
3
2.2.4.6 Probe labelling, “Random Priming” and hybridization …………….. 52
2.2.4.7 Reverse transcription and Polymerase Chain Reaction (PCR):
Semi-quantitative RT-PCR ………………………………………..... 52
2.2.5 Transfection analysis ………………………………………………………….. 53
2.2.5.1 Stable transfection protocol “Effectene” …………………………… 53
2.2.5.2 Stable transfection by Calcium-Phosphate co-precipitation ………... 54
2.2.6 DNA staining with DAPI and SR 101 for flow cytometry and cell cycle
analysis ………………………………………………………………………... 55

III. RESULTS ……………………………………………………………………………... 56
3.1 Analysis of HPV-positive cell lines after HDAC inhibition ………………………….... 56
3.1.1 HDAC inhibitors induce pRb degradation only in the presence of HPV
oncogenes …………………………………………………………………………... 56
3.1.2 HPV E7 is selectively responsible for the degradation of pRb in the presence
of HDAC inhibitors ………………………………………………………………… 58
3.1.3 Degradation releases negatively regulated genes from pRb suppression ……. 59
3.2 The role of HPV E7 in pRb degradation by SB ……………………………………….. 61
3.3 pRb degradation induced by HDAC inhibitors is mediated by the 26S proteasome …… 64
3.4 Inhibitor of serine proteases, TLCK, modifies E7 proteins and inhibits its binding
to pRb …………………………………………………………………………………… 65
3.4.1 Tretament of HPV16 E7-immortalised keratinocytes with TLCK also rescues
pRb from its SB mediated degradation ………………………………….................. 68
3.5 HDAC inhibitors SB and TSA upregulate the cyclin dependent kinase inhibitor p21
and induces G1 arrest in HeLa cells ………………………………………………………... 70
3.5.1 Cell cycle analysis after HDAC inhibition …………………………………... 71
3.5.2 TLCK interferes with sodium butyrate induced G1 arrest …………………… 72
3.6 HDAC inhibitors SB and TSA induce apoptosis only in the presence of HPV ……….. 74
3.6.1 SB and TSA induce apoptosis in HPV positive cells via the propapoptotic
gene p73 …………………………………………………………………………….. 77
3.6.2 TLCK treatment leads to a suppression of the H