dc.contributor.author
Gratchev, Alexei
dc.date.accessioned
2018-06-07T21:45:20Z
dc.date.available
2001-11-06T00:00:00.649Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/8381
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-12580
dc.description
Title 1
Acknowledgments 7
Summary 8
1 Introduction 10
1.1 Colon carcinoma 10
1.1.1 Genetic alterations during colon carcinogenesis 10
1.1.2 Mucinous colon carcinoma 11
1.2 DNA methylation 13
1.2.1 Function of DNA methylation in prokaryotes 14
1.2.2 Differences of DNA methylation between eukaryotes and prokaryotes 14
1.2.3 Regulation of DNA methylation in eukaryotic cell 15
1.2.4 CpG islands 16
1.2.5 Regulation of transcription by methylation 17
1.3 Role of DNA methylation in cancer 18
1.3.1 Methylation dependent mutations 18
1.3.2 Overall decrease of DNA methylation in cancer cells 19
1.3.3 Regional hypermethylation in cancer 20
2 Objectives of this work 23
3 Materials and Methods 24
3.1 Tissue samples 24
3.1.1 Preparation of the sections for microdissection 24
3.1.2 Microdissection 24
3.2 Cell lines 25
3.3 Chemicals and products for molecular biology 25
3.3.1 Buffers and mediums 25
3.3.2 Kits 26
3.3.3 Restriction enzymes 26
3.3.4 DNA modifying enzymes 26
3.3.5 Primers and oligonucleotides 27
3.3.5.1 Primers for PCR 27
3.3.5.1.1 MUC2 cDNA 27
3.3.5.1.2 MUC2 promoter region 27
3.3.5.1.3 MUC2 first intron region 27
3.3.5.1.4 Bisulphite modified MUC2 promoter sequence 28
3.3.5.1.5 DNMT2 methyltransferase (Acc. Nr.: AF045888) 28
3.3.5.1.6 Maintenance Methyltransferase DNMT1 (Acc. Nr.: X63692) 29
3.3.5.1.7 p21waf (Acc. Nr.: U03106) 29
3.3.5.1.8 PDH (Acc. Nr.: D90086) 29
3.3.5.2 Standard sequencing primers 29
3.3.5.3 Primers for Single Nucleotide Primer Extension (SNuPE) 30
3.3.6 Polymerase chain reaction (PCR) 30
3.3.6.1 PCR for MUC2 expression analysis 31
3.3.6.2 MUC2 promoter region 31
3.3.6.3 PCR of the first intron of MUC2 31
3.3.6.4 PCR of bisulphite treated DNA isolated from cell lines 32
3.3.6.5 PCR of the bisulphite treated DNA from sections 33
3.3.6.6 PCR of DNMT2 methyltransferase 34
3.3.6.7 PCR of DNMT1 methyltransferase 35
3.3.6.8 PCR of bisulphite treated plasmid DNA 35
3.3.6.9 PCR for site directed mutagenesis in the promoter region 36
3.3.7 Probes for Northern and Southern blots 36
3.3.7.1 Northern blot of MUC2 36
3.3.7.2 Southern blot of MUC2 36
3.3.7.3 Oligonucleotide of first 30 bp of MUC2 gene 37
3.3.7.4 18S rRNA 37
3.3.7.5 p21 probe 37
3.3.7.6 Luciferase gene probe 37
3.3.8 Plasmid vectors 37
3.3.9 Bacteria 38
3.3.10 Genomic library 38
3.4 Methods of lambda phage analysis 38
3.4.1 Plating the phage 38
3.4.2 Titration of phage stock 38
3.4.3 PCR based screening of l DNA library 38
3.4.4 High titer stock preparation 42
3.4.5 Large scale phage DNA preparation 42
3.5 Plasmids preparations 43
3.5.1 Transformation of E.coli 43
3.5.2 Minipreps 44
3.5.3 Midipreps 44
3.5.4 Isolation of DNA fragments from the agarose gel 44
3.5.5 Cloning of the DNA fragment into the plasmid vector 45
3.5.6 Cloning of the PCR product in TOPO TA Cloning vector 45
3.6 Analysis of genes expression 45
3.6.1 Total RNA isolation 45
3.6.2 Northern blot 46
3.6.2.1 Preparation of the gel 46
3.6.2.2 Transferring the RNA on the membrane 46
3.6.2.3 Labelling of the probe 46
3.6.2.4 Hybridisation of the membrane 46
3.6.3 RT-PCR 47
3.6.3.1 cDNA synthesis 47
3.7 Analysis of DNA methylation 48
3.7.1 Southern blot 48
3.7.2 Analysis of methylation by bisulphite sequencing 49
3.7.3 Analysis of methylation by SNuPE 49
3.7.4 Inhibition of methylation with 5-aza-2'-deoxycytidine 52
3.8 Reporter analysis of the promoter activity 52
3.8.1 In vitro methylation 52
3.8.2 Transfection of cells 52
3.8.3 Luciferase and b-galactosidase assays 52
3.8.4 Normalising the transfection with DOT blot hybridisation 53
4 Results 54
4.1 Isolation and characterisation of the MUC2 promoter 54
4.1.1 MUC2 5' region containing lambda clone 54
4.1.2 Sequencing and analysis of the promoter region 60
4.1.3 Analysis of the regulation of the MUC2 promoter 62
4.2 Methylation of the promoter in different cell lines 66
4.2.1 MUC2 promoter methylation in MUC2 expressing and non expressing cell
lines 66
4.2.2 Analysis of MUC2 promoter methylation by bisulphite sequencing 71
4.2.3 Activation of MUC2 expression by inhibition of methylation 73
4.3 MUC2 promoter methylation in cloned cells 73
4.3.1 Inhibition of methylation and selection of clones 75
4.3.2 Methylation of the MUC2 promoter in obtained clones 77
4.3.3 Gene expression alterations in clones 80
4.4 Influence of the site-specific methylation influence on the promoter
activity 82
4.4.1 Influence of methylation on the promoter activity in different cell
lines 82
4.4.2 Analysis of the effect of mutations at CpG sites #5 and #8 84
4.4.3 Analysis of possible de novo methylation or demethylation of the
transfected plasmid 85
4.5 MUC2 promoter methylation in tissues 86
4.5.1 Application of microdissection 86
4.5.2 Analysis of MUC2 promoter methylation in tissue 87
4.5.3 MUC2 promoter methylation in isolated goblet cells 88
5 Discussion 90
5.1 MUC2 promoter sequence analysis 90
5.2 Regulation of MUC2 expression in cell lines 90
5.3 Regulation of MUC2 expression in malignant and normal colonic tissues 94
5.4 The role of methylation in the development of the mucinous carcinoma 97
6 Reference List 99
Curriculum Vitae 109
dc.description.abstract
The development of the mucinous phenotype of colorectal carcinoma is
associated with the overexpression of the main intestinal mucin MUC2 while in
the nonmucinous carcinomas this gene is suppressed. This difference between
the two phenotypes as well as other differences, such as frequency of p53 and
Ki-ras mutations suggested that development of mucinous and nonmucinous
tumours occurs along two distinct genetic pathways. To clarify the details of
these pathways, the mechanism of MUC2 gene expression regulation was
investigated. In the present work it is shown, that DNA methylation plays an
essential role in the regulation of MUC2 promoter activity. The detailed
analysis of this mechanism showed, that MUC2 expression in vitro is determined
by overall MUC2 promoter methylation and particularly by the status of a CpG
site following the TATA box. Normal human goblet cells and mucinous colorectal
carcinomas, which both strongly express MUC2, exhibit average methylation of
about 50% at every CpG site of the MUC2 promoter. By contrast, in normal
columnar cells, which do not express MUC2, and in nonmucinous carcinomas
methylation of the MUC2 promoter was nearly 100%.
These results show that the methylation patterns of MUC2 promoter in mucinous
and nonmucinous carcinomas correspond to that of normal goblet and columnar
cells, respectively. These data are compatible with the hypothesis that the
development of mucinous colorectal carcinoma is not caused by a faulty
regulation of the MUC2 gene but is a result of normal goblet cell
differentiation process on which the carcinogenic lesions are superimposed.
de
dc.description.abstract
Die Entwicklung des mucinösen Phänotyps des Coloncarcinoms ist hauptsächlich
mit der Überexpression des intestinalen Mucin MUC2 verbunden, während in den
nichtmucinösen Karzinomen dieses Gen unterdrückt ist. Der Unterschied zwischen
diesen zwei Phänotypen als auch weitere Unterschiede, solche wie die
Häufigkeit von p53 und Ki-ras Mutationen legen nahe, dass die Entwicklung von
mucinösen und nichtmucinösen Karzinomen über verschiedene genetische Wege
abläuft. Um Einzelheiten dieser Wege aufzuklären wurde der
Regulationsmechanismus der MUC2-Genexpression untersucht. In der vorliegenden
Arbeit wird gezeigt, dass die DNA-Methylierung eine wesentliche Rolle bei der
Regulation der MUC2-Promotoraktivität spielt. Die Detailanalyse dieses
Mechanismus zeigt, dass die MUC2-expression in vitro durch die generelle
MUC2-Promotormethylierung und insbesondere eines CpG hinter der TATA-Box
bestimmt wird. Normale menschliche Becherzellen und mucinöse Coloncarcinome,
welche beide MUC2 in hohem Maß exprimieren, zeigen etwa 50% Methylierung des
CpG-Areale des MUC2 Promotors. Im Vergleich dazu beträgt die Methylierung des
MUC2-Promotors in Zylinderepithelzellen, die MUC2 nicht exprimieren, und in
nicht mucinosen Karzinomen annähernd 100%.
Die Resultate zeigen, dass der Methylierungsgrad des MUC2-Promotors in
mucinösen und nicht mucinösen Karzinomen dem von normalen Becher- und
Zylinderepithelzellen entspricht. Die Ergebnisse stimmen mit der Hypothese
überein, dass die Entwicklung von mucinösen Coloncarcinomen nicht durch eine
Regulationsstörung im MUC2-gen bedingt ist, sondern eine Folge normaler
Becherzelldifferenzierung die von der Karzinogenen Veränderung überlagert ist.
de
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::570 Biowissenschaften; Biologie
dc.title
Regulation of expression of the intestinal MUC2 gene in vitro and in vivo
dc.contributor.firstReferee
Prof. Dr. Christoph Hanski
dc.contributor.furtherReferee
Prof. Dr. Eberhard Riedel
dc.date.accepted
2001-08-09
dc.date.embargoEnd
2001-11-08
dc.identifier.urn
urn:nbn:de:kobv:188-2001002133
dc.title.translated
In vivo und in vitro Regulation der Expression des intestinalen MUC2 Gens
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000000541
refubium.mycore.transfer
http://www.diss.fu-berlin.de/2001/213/
refubium.mycore.derivateId
FUDISS_derivate_000000000541
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access