Comparative translational, transcriptional, and functional profiling of clear cell and papillary renal cell carcinoma [Elektronische Ressource] / written by Julia Diegmann

Julia Diegmann

Sujets

• Gesundheit

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2006
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

COMPARATIVE
TRANSLATIONAL, TRANSCRIPTIONAL, AND FUNCTIONAL PROFILING
OF
CLEAR CELL AND PAPILLARY RENAL CELL CARCINOMA



Academic Dissertation (Doctor rerum naturalium)
at the
Faculty of Biology
of the
Friedrich-Schiller University, Jena, Germany




Written by
Certified Biologist Julia Diegmann
born 20th of February, 1977 in Fulda






















Gutachter:

1. PD Dr. von Eggeling
2. Prof. Dr. Pool-Zobel
3. Prof. Dr. Hartmann
Tag der mündlichen Prüfung: 21.07.2006
Tag der öffentlichen Verteidigung: 06.11.2006


























Mit dem Wissen wächst der Zweifel.

Johann Wolfgang von Goethe

Outline
1 Abstract ..........................................................................................................1
2 Introduction.....................................................................................................2
2.1 Expression Profiling and Marker Gene Discovery.........................................................2
2.2 Renal Cell Carcinoma .........................................................................................4
2.2.1 Pathological Classification of Renal Cell Carcinoma ..............................................4
2.2.2 Marker Genes and Pathways in Renal Cell Carcinoma ............................................5
2.2.3 Immune Escape Mechanisms of Renal Cell Carcinoma ............................................7
3 Aims of This Study .............................................................................................9
4 Materials and Methods ...................................................................................... 12
4.1 Structural Overview of Experiments ..................................................................... 12
4.2 Materials ...................................................................................................... 13
4.2.1 Tissue Samples ......................................................................................... 13
4.2.2 Cell Lines and Native Lymphocytes................................................................. 15
4.3 Methods ....................................................................................................... 15
4.3.1 Translational Profiling ................................................................................ 15
4.3.2 Transcriptional Profiling.............................................................................. 18
4.3.3 Functional Profiling ................................................................................... 23
4.3.4 Functional Analysis of CD70 ......................................................................... 25
5 Results.......................................................................................................... 26
5.1 Translational Profiling ...................................................................................... 26
5.2 Transcriptional Profiling ................................................................................... 33
5.2.1 Confirmation of Differentially Expressed Genes ................................................. 37
5.3 Functional Profiling ......................................................................................... 41
5.4 Functional Analysis.......................................................................................... 47
5.5 Summary of Results ......................................................................................... 51
6 Discussion...................................................................................................... 53
6.1 Translational Profiling ...................................................................................... 53
6.2 Transcriptional Profiling ................................................................................... 55
6.3 Functional Profiling ......................................................................................... 57
6.4 Functional Analysis.......................................................................................... 60
7 Concluding Remarks......................................................................................... 63
8 Deutsche Kurzfassung....................................................................................... 64
9 References .................................................................................................... 65
9.1 List of Original Publications ............................................................................... 65
9.2 List of Cited Publications .................................................................................. 66
10 Equipment and Chemicals ................................................................................. 74
10.1 Equipment ............................................................................................... 74
10.2 Chemicals................................................................................................ 75
11 List of Tables and Figures.................................................................................. 78
11.1 List of Tables............................................................................................ 78
11.2 List of Figures........................................................................................... 78
12 Appendices .................................................................................................... 80

Abbreviations

aRNA amplified ribonucleic acid
Bp base pair
ccRCC clear cell renal cell carcinoma
cDNA copy desoxyribonucleic acid
CGH comparative genomic hybridization
2D-PAGE two-dimensional protein gel electrophoresis
DNA desoxyribonucleic acid
IHC immunohistochemistry
kDA kilo dalton
mM millimol
mTor mammalian target of rapamycin
no. number
PCR polymerase chain reaction
PHA-L phytohemagglutinin A
pRCC papillary renal cell carcinoma
Pmol picomol
RCC renal cell carcinoma
rmsCD70 recombinant soluble CD70
RNA ribonucleic acid
RT-PCR reverse transcription-polymerase chain reaction
SAM statistical analysis of microarrays
SELDI-TOF surface enhanced laser desorption ionisation – time of flight
TILs tumor-infiltrating lymphocytes
VHL von-Hippel Lindau gene
WHO world health organisation
µg Microgramm

Note: Gene names are written in italics
1 Abstract
1 ABSTRACT
The biology of renal cell carcinoma (RCC) remains poorly understood. RCC, which
accounts for up to 3% of all adult malignancies, is the most common neoplasm in
the adult kidney and has been increasing in incidence. These tumors are classified
into five main subtypes: clear cell, papillary, and chromophobe RCC, as well as
collecting duct carcinoma and oncocytoma. Of these, clear cell and papillary are
the two most frequently diagnosed subtypes and both arise from the proximal tube
of the kidney. Despite sharing a common origin, they are histologically and
genetically distinguishable and thus represent an optimal model system for
studying tumor-specific and tumor-subtype specific expression patterns. The aim of
this study was to identify potential marker genes for clear cell and papillary RCC,
assess their specificity and relevance for the tumor-subtypes, and thus contribute
to a better characterization of the tumor development and behavior of RCC.
To address these questions, this study combined translational and transcriptional
approaches to analyze the protein and gene expression of both tumor subtypes.
The translational approach identified tumor-subtype specific protein expression
patterns using SELDI-TOF technology (Surface Enhanced Laser Desorption Ionization
– Time of Flight). In this way, the first specific protein pattern for papillary RCC
was identified. The transcriptional approach also identified numerous genes having
tumor-subtype specific regulations by using cDNA-microarrays. Genes with high
potential to discriminate between normal and tumor tissue were further analyzed
with independent methods. Most strikingly, the gene CD70 was identified as being
specifically over-expressed in clear cell RCC, thus revealing a new, specific marker
gene. Finally, the gene expression data was bioinformatically analyzed to elucidate
the underlying biology of the gene regulations found. This revealed an
accumulation of apoptosis-inducing genes that are equally regulated in both RCC
subtypes.
At a first glance, the induction of apoptosis seems to be antithetical to tumor
survival. However, the induction of apoptosis in tumor-infiltrating lymphocytes,
which are frequently observed in RCC, would provide a strategy to escape immune
recognition. The group of apoptosis-inducing genes includes the gene CD70, which
was then further studied regarding its function for the tumors in cell co-culture
experiments. These experiments demonstrated the ability of CD70 to induce