Proteomics of acute myeloid leukemia: [Elektronische Ressource] : cytogenetic risk groups differ specifically in their proteome, interactome and posttranslational protein modifications / vorgelegt von Mumtaz Yaseen Balkhi

Mumtaz Yaseen

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Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 41
• Poids de l'ouvrage: 5 Mo

Extrait

Aus der Medizinischen Klinik und Poliklinik III-Großhadern-der
Ludwig-Maximilians-Universitäts München,
Director: Prof. Dr. Wolfgang Hiddeman





Proteomics of Acute Myeloid Leukemia: Cytogenetic Risk Groups
Differ Specifically in their Proteome, Interactome and
Posttranslational Protein Modifications



Dissertation
zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München



Vorgelegt von
Mumtaz Yaseen Balkhi
aus
Soura-Srinagar, India
2007





From the Department of Internal Medicine III,
Ludwig-Maximilians-University, Munich
Director: Prof. Dr. Wolfgang Hiddeman





Proteomics of Acute Myeloid Leukemia: Cytogenetic Risk Groups
Differ Specifically in their Proteome, Interactome and
Posttranslational Protein Modifications





Thesis
Submitted for a Doctoral degree in Human Biology
at the faculty of Medicine
Ludwig-Maximilians-University, Munich



Submitted by
Mumtaz Yaseen Balkhi
From
Soura-Srinagar, India
2007









Mit Genehmigung der Medizinischen Fakultät
der Universität München



Berichterstatter: Prof. Dr. Stefan K Bohlander



Mitberichterstatter: Prof. Dr. H. G. Klobeck
Prof. Dr. B. Eimmerich


Mitbetreuung durch den
promovierten Mitarbeiter: PD. Dr. med. Gerhard Behre



Dekan: Prof. Dr. med. D. Reinhardt


Tag der mündlichen Prüfung: 12. 06. 2007






With permission from the Faculty of Medicine
University of Munich


Supervisor/Examiner: Prof. Dr. Stefan K Bohlander




Co-Examiners: Prof. Dr. H. G. Klobeck
Prof. Dr. B. Eimmerich





Co-Supervisor: PD. Dr.med.Gerhard Behre

Dean: Prof. Dr. med. D. Reinhardt

Date of Submission: 25.10. 2006

Date of Oral Exam: 12. 06. 2007

























Dedicated To My Father Mohd Yaseen Balkhi
&
Mother Shahzada Yaseen
Table of Contents
Abbreviations IV
1 Introduction............................................................................................................1
1.1 Acute myeloid Leukemia…………................................................................1
1.2 Chromosomal translocations in AML............................................................4
1.2.1 The core binding factor complex……………………………...………......6
1.2.2 Acute promyelocytic leukemia....................................................................8
1.2.3 Acute myeloid leukemia with 11q23 (MLL) abnormalities........................9
1.3 Survivin.......................................................................................................10
1.4 Self-renewal and AML................................................................................14
1.5 Proteomics based on mass spectrometry..................................................... 15
1.5.1 Post-translational modifications (PTMs) ................................................... 18
1.6 Aim of the study ........................................................................................... 20
2 Materials and methods................................................................................. 21
2.1 Material ....................................................................................................... 21
2.1.1 Mammalian cell line:.................................................................................. 21
2.1.2 Cell culture ................................................................................................. 21
2.1.3 Immunoblots............................................................................................... 22
2.1.4 Antibody..................................................................................................... 22
2.1.5 Plasmid constructs and transient transfections........................................... 22
2.1.6 Chemicals ................................................................................................... 22
2.2 Methods....................................................................................................... 23
2.2.1 AML patient samples ................................................................................. 23
2.2.2 Two dimensional gel electrophoresis and DIGE ....................................... 24
2.2.3 Statistical analysis ...................................................................................... 25
2.2.4 MALDI-TOF mass spectrometry............................................................... 25
2.2.5 Transient transfections using LipofectAMINE plus and reporter assays
firefly and renilla luciferase ................................................................................ 26
2.2.6 shRNA and flow cytometric analysis......................................................... 27
I2.2.7 Quantitative real-time PCR analysis .......................................................... 29
2.2.8 Immunoblot analysis .................................................................................. 29
2.2.9 Electrophoretic Mobility-Shift Assay (EMSA) ......................................... 30
3 Results............................................................................................................. 31
3.1 Mass spectrometry based identification of proteins from different AML
cytogentic groups………………………………………………………………31
3.2 Proteins identified as distinguished targets of AML cytogenetic
groups.................................................................................................................40
3.3 Validation of differential expression of hnRNPA2/B1, casein kinase 1
alpha, prohibitin and HSC70............................................................................... 45
3.4 Survivin identified as a distinguished target of t(8;21) AML...................... 47
3.5 AML1/ETO induces survivin expression ................................................... 48
3.5.1 AML1-ETO activates basal transcription of the survivin promotor.......... 51
3.5.2 AML1-ETO binds to the survivin promoter .............................................. 53
3.6 Knockdown of survivin expression overcomes AML1-ETO mediated
inhibition of C/EBP α autoregulation................................................................... 55
3.7 Repression of survivin expression by shRNA restores C/EBP α expression
inhibited by AML1/ETO..................................................................................... 57
3.8 Repression of survivin expression overcomes granulocytic differentiation
block induced by AML1/ETO in human CD34+ cells ....................................... 63
3.9 Cytogenetic groups differ in their protein interaction networks .................. 66
3.10Cytogenetic groups showed significant differences in their protein
expression patterns .............................................................................................. 71
3.11DIGE method identifies significant quantitative variation of expression
pattern between t(15;17)- and inv(16)-AML ...................................................... 72
3.12Calreticulin, hnRPH1 and hnRNPA2/B1 showed a difference in their
posttranslational modifications between cytogenetic risk groups ...................... 74
4 Discussion ....................................................................................................... 84
5 Summary ......................................................................................................... 91
II6 Zusammenfassung........................................................................................... 93
7 Reference......................................................................................................... 96
8 Acknowledgement......................................................................................... 109
9 Appendix .......................................................................................................