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Protein dynamics in responder and non-responder solid tumor xenografts during oncolytic viral therapy [Elektronische Ressource] / vorgelegt von Thu Ha Le

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Bayerische Julius-Maximilians-Universität zu Würzburg Fakultät für Biologie Lehrstuhl für Mikrobiologie Protein dynamics in responder and non-responder solid tumor xenografts during oncolytic viral therapy Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Thu Ha Le aus Vietnam Würzburg, 2008 Eingereicht am: ..................................................................................................................... Mitglieder der Promotionskommission: Vorsitzender: ......................................................................................................................... Gutachter : ............................................................................................................................. Gutachter: .............................................................................................................................. Tag des Promotionskolloquiums: ........................................................................................ Doktorurkunde ausgehändigt am: ........................................................................................ Supervisor 1: (Prof. Dr. A. A.

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Publié par
Publié le 01 janvier 2009
Nombre de lectures 46
Langue Deutsch
Poids de l'ouvrage 7 Mo

Bayerische Julius-Maximilians-Universität zu Würzburg
Fakultät für Biologie
Lehrstuhl für Mikrobiologie


Protein dynamics in responder and non-responder solid
tumor xenografts during oncolytic viral therapy








Dissertation
zur Erlangung des naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg


vorgelegt von
Thu Ha Le
aus Vietnam

Würzburg, 2008
Eingereicht am: .....................................................................................................................

Mitglieder der Promotionskommission:
Vorsitzender: .........................................................................................................................
Gutachter : .............................................................................................................................
Gutachter: ..............................................................................................................................

Tag des Promotionskolloquiums: ........................................................................................

Doktorurkunde ausgehändigt am: ........................................................................................


















































Supervisor 1:
(Prof. Dr. A. A. Szalay, Lehrstuhl für Mikrobiologie)









Supervisor 2:

(Prof. Dr. F. Grummt, Lehrstuhl für Biochemie)























Eidesstattliche Erklärung

Hiermit versichere ich, dass ich die vorliegende Dissertation selbständig und nur mit den
angegebenen Hilfsmitteln und Quellen angefertigt habe.

Ich erkläre außerdem, dass diese Dissertation weder in gleicher noch in anderer Form bereits
in einem anderen Prüfungsverfahren vorgelegen hat.

Neben dem akademischen Grad „Master-MD-Uni.“ habe ich keine weiteren akademischen
Grade erworben oder zu erwerben versucht.



Würzburg, den ……………….. ………………………………...
(Thu Ha Le)






ACKNOWLEDGEMENT
At this point I would like to take the chance to express my gratitude to all those who
gave me the possibility to complete this thesis that took place at the department of
stmicrobiology, Bio center of the University of Wuerzburg, Germany, from October 1 of 2004
until December of 2008 under supervision of Prof. Dr. A. A. Szalay and Prof. Dr. F. Grummt.
I special thanks to
Prof. Dr. A. A. Szalay for providing working environment and for representing this
dissertation at the faculty of biology. I am deeply indebted to you for your kindness for giving
a very interesting thesis as well as stimulating suggestions, kindly recommendation, financial
support of scientific projects and for your continuous encouragement.
Prof. Dr. F. Grummt for his interest on my project and for proofreading this work. I
particularly thank you for encouragement helped me in all the time of research for and writing
of this thesis, looked closely at the final version of the thesis for English style and grammar,
correcting both and offering suggestions for improvement.
Dr. Andrea Spory from Microbiology department of Wuerzburg University for her
kindly technical assistance of two-dimensional electrophoresis.
Dr. Jörg Bernhardt from Institute of Microbiology in Greifswald University for his
meticulous assistance in instructing in analysis a differential protein expression by using 2D-
Software and Dr. Brigit Voigt from Institute of Microbiology in Greifswald University for her
responsible assistance in protein sequencing
Dr. Benedikt Brors from German cancer research centre for computative tumor protein
analysis.
I was delighted to interact with Dr. Elisabeth Hofmann by her help and having her as
my co-advisor, specially thank for her thorough assistance and for illuminative discussions.
My colleagues in the laboratory Christine Horchbashek, Chiristina Tietze, Stephanie
Weibel, Carolin Seubert, Victoria Raab, Andrea Worschech, Ulrike Geißinger, Dr. Jochen
Stritzker, Dr. Ivaylo Gentscher, Johanna Langbein, Ulrike Donat, Julia Sturm, Yvonne
Dombrowski, Anja Sauer, Kerstin Richte, Claudia Stühler for their sedulous helpfulness and
the comfortable working atmosphere, for advice in word and deeds and cheerful hours, the
staff members of the chair of microbiology, especially Monika Goetz for kindly support
working facilities, as well as the personnel of the chair of biochemistry, specifically to Anneli
Kießling for her organizational talent and aid.
All members of staff of Genelux company, named Tony Yo, Qian Zhang, Nanhai
Chen, Alexa Frentzen, Antonietta Jimenez Pearson, Terry Trevino, Camha Hoang, Alison Estrada, Shahrokh Shabahang, Andrea Feathers, Tom Hagood, Valere Rollings, Albert Röder,
Ron Simus and Daniela for financial support of scientific projects as well as kindly, helpful
assistance during the past 4 years.
I would regret my doctoral years at Wuerzburg University if I did not join MOET and
DAAD fellowships. Joining these fellowships was not only a turning point in my life, but also
a wonderful experience.
Sincere thanks to my mother who always supported me and to my son and husband in
Vietnam who had to live without me during the past years of my doctorade, whose patient
love enabled me to complete this work. I am deeply grateful for their true understanding and
trust, their help and support and much.


Contents
ABSTRACT..........................................................................
1. INTRODUCTION.................................................................................................. 1
1 1.1. Vaccinia virus as a representative oncolytic virus………………………...
1.2. Vaccinia virus biology………………………………………………………. 2
1.2.1. Vaccinia virion structure and life cycle..................................................... 2
1.2.2. Interactions with host cells ……… ……… … ……… ……… ……… …… ……. 3
1.3. Genetic engineering for tumor selective replication………………............ 5
1.4. Mechanisms of tumor escape………………………………………………. 7
1.5. Proteomic strategies and their application cancer research……………... 10
1.5.1. Cancers ……………………………………… …………………………… …….. 10
1.5.2. Cancer proteomic …………………………… ……. 10
1.5.3. Principles and technological platforms …… ……… ……… ……… …… …... 11
1.5.3.1. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE ) … ... 12
1.5.3.2. Mass spectrometry ………………………………………………………. ... ... 12
1.5.4. Proteome analysis of human cancer ……… ……… ……… ……… …… ……. 15
1.5.4.1. Proteome analysis of human breast cancer …………………………… …. 15
1.5.4.2. Proteome analysis of human colon cancer ….. 16
1.5.4.3. Proteome analysis of human prostate cancer …………………………….. 18
1.5.5. Proteomics analysis the differentially expressed of cancer cells infected
with virus …… ……… ……… ……… …… ……… ……… ……… …… ……… … 19
1.5.5.1. Modification of apoptotic pathways in the host cell …………………….. 21
1.5.5.2. Modification in the cytoskeleton structure of the host cell ……… ……... 23
1.5.5.3. Modification of cell host intracellular signaling pathways ……… … ….. 24
1.5.5.4. Modif cell host metabolic pathways ……………………………. 26
AIMS OF THE STUDY............................................................................................. 29
30 2. MATERIALS AND METHODS...........................................................................
2.1 MATERIALS........................................................................................................ 30
2.1.1 Equipments..................................................................................................... 30
32 2.1.2 Reagents, solutions and media.......................................................................
2.1.2.1 Reagents................................................................................................... 32
2.1.2.2 Solutions and media................................................................................. 34
2.1.3 Cell lines…………………………………………………………………….. 35
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35 2.1.4 Recombinant vaccinia virus VACV GLV-1h68.............................................
2.1.5 Antibodies........................................................................................................ 36
2.2 METHODS............................................................................................................ 38
38 2.2.1 Cell biological methods..................................................................................
2.2.1.1 Cell culture.............................................................................................. 38
2.2.1.2 Cell quantification ………………………… …………………………… ……. 38
2.2.1.3 Virus infection ………… ……. 39
40 2.2.2 Gene expression....
2.2.2.1 Fluorescent microscopy........................................................................... 40
2.2.2.2 Western blot............................................................................................ 40
41 2.2.3 Trypan blue staining.......................................................................................
2.2.4 Titration of vaccinia virus by plaque assay................................................... 42
2.2.5 Protein analysis…………………………………………………………….. 43
2.2.5.1 Sample preparation and protein solubilisation....................................... 43
2.2.5.2 Protein precipitation............................................................................... 43
2.2.5.3 Determination of protein concentration by Bradford assay................... 44
2.2.5.4 Separation of proteins in one dimensional gel electrophoresis and
two-dimensional gel electrophoresis....................................................... 44
2.2.5.4.1 One dimensional gel electrophoresis.................................................... 44
2.2.5.4.2 Two dimensional gel electrophoresis................................................... 44
2.2.5.5 Computerized 2-D image anylysis............. 48
2.2.5.6 Mass spectrometry for protein identification.......................................... 50
3. RESULTS................................................................................................................ 53
3.1 Verification of marker expression VACV GLV-1h68................................... 53
3.1.1 GFP-fluorescence microscopy study......................................................... 53
3.1.2 Biochemical analysis of cell lysate............................................................ 58
3.1.2.1 Expression of GFP-RUC fusion proteins encoded in the VACV GLV-
1h68 infected cells …………………………… …………………………… … 58
3.1.2.2 Expression of β-galactosidase proteins in the VACV GLV-1h68
infected cells ……… ……… ……… ……… …… ……… ……… ……… …… … 59
3.1.2.3 Expression of β-glucuronidase proteins encoded by marker genes in
the tumor cells ………………………… …………………………… ………. 60
3.1.2.4 Expression of marker proteins of the tumor xenograft in mice …… …. 61
3.2 Growing characteristic of tumor cell lines non-infected and infected with
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65 VACV GLV-1h68.............................................................................................
3.3 Replication of VACV GLV-1h68 in the cell cultures.................................... 68
3.4 Enzyme-linked immunoabsorbent assays (ELISAs) using a ‘two-site’
71 Sandwich detection assay……………………………………………………
3.5 Two-dimensional gel electrophoresis profiles of VACV GLV-1h68
infected tumors and cells............................................................................... 74
3.5.1 Comparison of differential protein expression…………………………... 80
3.5.1.1 Comparison of differential protein expression in VACV GLV-1h68
un-infected and infected GI-101A cells …………………………… …………… 80
3.5.1.2 Comparison of differential protein expression in VACV GLV-1h68
un-infected and infected GI-101A tumors ………… 82
3.5.1.3 Comparison of differential protein expression in VACV GLV-1h68
un-infected and infected HT-29 cells … ……… ……… ……… …… ……… …… 83
3.5.1.4 Comparison of differential protein expression in VACV GLV-1h68
un-infected and infected HT-29 tumors ……………………………… …………. 83
3.5.1.5 Comparison of differential protein expression in VACV GLV-1h68
un-infected and infected PC-3 cells …………………………………… …………. 84
3.5.1.6 Comparison of differential protein expression in VACV GLV-1h68
un-infected and infected PC-3 tumors …… ……… ……… ……… …… ……… … 85
3.5.2 Comparison of regulated and modified reproducibly identified upon
86 VACV GLV-1h68 infections.........................................................................
4. DISCUSSION....................................................................................................... 101
4.1 VACV GLV-1h68 infection hijacking of the host translation apparatus…… 101
4.2 Alteration of cytoskeleton networks………………………………………… 105
4.3 Ubiqitin proteasome pathway (UPP) disorders in VACV GLV-1h68 infected
tumor cells…………………………………………………………………… 105
4.4 VACV GLV-1h68 alter stress response proteins…………………………… 107
109 4.5 Infection effect to anti-apoptosis pathways in cancer cells…………………
4.6 Infection effect to activated EGFR or Ras pathways……………………… 112
4.7 Infection effect to the hypoxic tumor environment………………………… 113
114 4.8 Chemokines in cell homing to cancer………………………………………...
5. REFERENCES....................................................................................................... 117
6. ABBREVIATIONS……………………………………………………................. 142
144 7. TABELS…………………………………………………………………………..
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T1- p1 Table 1. Differential expression proteins in GI-101A tumors………………...
Table 2. Differential expression proteins in HT-29 tumors………… T 2-p1
Table 3. Differential expression proteins in PC-3 tumors……………………. T3- p1
T4- p1 Table 4. Differential expression proteins in GI-101A cells……………………
Table 5. Differential expression proteins in HT-29 cells……………………… T5- p1
Table 6. Differential expression proteins in PC-3 cells……………………….. T6- p1
145 8. CURRICULUM VITAE……………………………………………....................

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