Stroma-leukaemic cell interactions [Elektronische Ressource] : analysis of stroma environment-induced effect on human acute myeloid leukaemic cells / Kallal Pramanik

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• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 24 Mo

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Stroma-leukaemic cell interactions: Analysis of stroma
environment-induced effect on human acute myeloid
leukaemic cells




Thesis submitted to the Julius-Maximilians-Universität, Würzburg,
Germany, towards fulfilment of the requirements for the Doctorate
degree





Bayerische Julius-Maximilians-Universität Würzburg, Germany
Faculty of Biology





Kallal Pramanik
Nadia, West Bengal, India

Würzburg, Germany
November 2006
Eingereicht am:……………………………………………………………


Mitglieder der Promotionskommission:

Vorsitzender:.............................................................................................

Gutachter: ………………………...................…...….. Prof. Dr. A. Müller
Gutachter: ……………………...................……….......Prof. Dr. U. Scheer


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

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

ii
DECLARATION








I hereby declare that the submitted dissertation was completed by myself and no
other, and I have not used any sources or materials other than those enclosed.

Moreover, I declare that the following dissertation has not been submitted further in
this form and has not been used for obtaining any other equivalent qualification in
any other organization. Additionally, other than this degree I have not applied or will
not attempt to apply for any other degree, title or qualification in relation to this work.






Kallal Pramanik
Date:
Place: Würzburg
iii
ACKNOWLEDGEMENTS


I want to express my deep sense of gratitude to my supervisor Prof. Dr. Albrecht Müller for
his meticulous guidance and unwavering help and encouragement during the course of this
study. I thank my thesis committee members Prof. Dr. Manfred Schartl and Prof. Dr. Ulrich
Scheer for their attention. I thank Prof. Dr. Manfred Gessler for his advices and support.
I acknowledge Prof. Dr. Ulf Rapp for providing the facilities during the course of the present
study.
I will like to thank my research collaborators, Prof. Dr. Reinhardt Henschler and Prof. Dr. Axel
Greiner, for their advices and assistance. I will take this opportunity to thank MSZ research
and journal seminar club. My special thanks to Dr. Joe Fensterle, Dr. Mathias Becker and Dr.
Hannes Dexler for their constructive comments and suggestions.
I take this opportunity to thank all the members of this institute for their help and cooperation.
Dr. Wolf Hans Thomas and Mr. Reinnhold Krug deserve special thanks for helping me in
irradiation facility.
I was lucky to have excellent lab mates, who have made the lab an enjoyable place to work. I
am especially thankful to Andrea Brandt for excellent technical support. I thank Vroni, Nikos,
Timo, Vlada, Duttu, Sandra and Nadine for the valuable discussions and constructive
criticisms of my work among numerous other things. Thanks to my previous labmates
Michael, Nikki, Suzana, Juergen and Carolin for their help during my initial days at the lab. I
acknowledge timely help and assistance from Ewald and Rosemarie.
I would like to thank all my long standing friends, who helped me in innumerable ways, both
in my ‘academic’ and ‘non-academic’ ventures. Thanks to Kajal, Sunit, Aditya and Chandan
for standing by me through thick and thin. I owe my thanks to Amiya, Naresh, Jayashree, Biju,
Anu and Arnab for providing me some quality time in their company.
I am deeply indebted to my parents and parents-in-law, whose blessings and inspirations
are always with me. Last but not the least; I thank my wife, Aditi, for her advices, support and
encouragement in every step of my life.
Financial support from Graduate College, GRK 639 639 “Molekulare und Strukturelle
Grundlagen der Tumorinstabilität“, during the course of study is duly acknowledged.



iv Content

1. SUMMARY.........................................................................................................................1
2. ZUSAMMENFASSUNG .....................................................................................................3
3 INTRODUCTION.................................................................................................................5
3.1 Acute myeloid leukaemia – The disease...................................................................5
3.2 Acute Myeloid leukaemia: The biology ...............................................................6
3.2.1 Acute myeloid leukaemia and translocation t(8;21) ................................................8
3.3 Cell cycle regulatory proteins and their role in cell proliferation and cell cycle
arrest ...............................................................................................................................10
3.4 Response of AML cells to differentiation-inducing agents..............................11
3.4.1 Targeting AML with antibodies.............................................................................14
3.5 Tumour cells and microenvironment background ...........................................15
3.5.1 Reprogramming of tumour in embryonic environment ...................................16
3.6 Sites of embryonic and adult haematopoiesis and haematopoietic stroma...17
3.6.1 Dorsal aorta stromal (DAS) 104-4 and DAS 104-8 cell lines ................................19
3.6.2 Yolk sac endodermal (YSE) and Yolk sac mesodermal (YSM) cell lines..............20
3.6.3 The foetal liver AFT 024 cell line..........................................................................20
3.7 Experimental strategy ..............................................................................................21
4 RESULTS .........................................................................................................................23
4.1 Myeloid marker expression following coculture of human AML cell line, Kasumi-
1, on murine stromal cell lines ......................................................................................23
4.2 DAS 104-4 coculture-induced acquisition of myeloid differentiation markers of
Kasumi-1 cells ................................................................................................................24
4.3 DAS 104-4 stroma can induce myeloid differentiation of patient-derived primary
M2-AML cells ..................................................................................................................26
4.4 Effect of cocultivation on KG-1 AML cells ..............................................................28
4.5 Coculture-induced change in proliferation properties of AML cells.....................31
4.6 DAS 104-4 induces decrease in total cell number and reduction in primitive
colony forming cell pool of cocultured AML cell population ......................................33
4.7 Effects of direct and indirect coculture...................................................................36
4.8 Effect of fixed DAS 104-4 in reduction of proliferation and induction of
differentiation of cocultured Kasumi-1 cells ................................................................39
4.8.1 Effect of fixed stroma on differentiation ................................................................40
4.9 Effect of DAS 104-4 stroma on Kasumi-1 AML1-ETO expression .........................41
4.9.1 DAS 104-4 induced reduction in AML1-ETO transcript level of cocultivated
Kasumi-1 cells ..............................................................................................................41
4.9.2 Stroma-AML contact-dependent down-regulation of AML1-ETO transcription .....42
4.10 CD44 ligation-induced differentiation of AML cells..............................................42
4.10.1 Expression of CD44 on Kasumi-1, KG-1 and NB-4 cells ....................................43
4.10.2 CD44-ligating antibody, A3D8 mediated differentiation of AML cells ..................43
4.11 Gene expression analysis of critical mediators of proliferation and myeloid
differentiation .................................................................................................................45
4.11.1 PU.1 ..................................................................................................................45
4.11.2 RUNX1 (AML1)..................................................................................................46
4.11.3 C/EBPα .............................................................................................................46
4.11.4 p16 (INK4A).......................................................................................................47
4.11.5 p21(WAF) ..........................................................................................................48
4.12 AML1-ETO siRNA decreases proliferation and induces myeloid differentiation
of cocultured Kasumi-1 cells.........................................................................................50
4.12.1 siRNA transfection efficiency ...................