Rolle der Ras-Signaltransduktion in der Pathophysiologie von myeloischen Leukämien und potentielle Effektivität von Inhibitoren der Ras-Signaltransduktionskaskade gegenüber menschlichen Tumoren [Elektronische Ressource] / von Michael Alexander Morgan

Reuter

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2003
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Rolle der Ras-Signaltransduktion in der Pathophysiologie von myeloischen Leukämien
und potentielle Effektivität von Inhibitoren der Ras-Signaltransduktionskaskade
gegenüber menschlichen Tumoren.




Von dem Fachbereich Chemie
der Universität Hannover
zur Erlangung des Grades einer

DOKTOR DER NATURWISSENSCHAFTEN
Dr. rer. nat.

Genehmigte Dissertation
von


MICHAEL ALEXANDER MORGAN
Geboren am 15.07.1967 in Watertown, New York, USA
2003


Role of Ras signaling in hematological malignancies and the potential role of inhibitors
of the Ras signaling cascade as anti-cancer agents.






Thesis to obtain the grade of
DOCTOR RERUM NATURALUM
(Dr. rer. nat)
of the University of Hannover

Specialities : Biochemistry and Molecular Biology






MICHAEL ALEXANDER MORGAN
Hannover 2002




Referent: Prof. Dr. rer. nat. W. Müller
Korreferent: Prof. Dr. med. A. Ganser/Prof. Dr. med. C.W.M. Reuter
Tag der Promotion: 19 Dezember 2002




























We dance round in a ring and suppose,
But the Secret sits in the middle and knows.
Robert Frost





Is my understanding only blindness to my own lack of understanding?
It often seems so to me.

Ist mein Verständnis nur Blindheit gegen mein eigenes Unverständnis?
Oft scheint es mir so.
Ludwig Wittgenstein





For reasons of priority, parts of these results have been published in :
Reuter CWM, Morgan MA, and Bergmann L (2000). Targeting the Ras signaling
pathway: A rational, mechanism-based treatment for hematological malignancies? Blood;
96: 1655-1669.

Morgan MA, Dolp O, and Reuter CW (2001). Cell-cycle-dependent activation of
mitogen-activated protein kinase kinase (MEK-1/2) in myeloid leukemia cell lines and
induction of growth inhibition and apoptosis by inhibitors of RAS signaling. Blood;97:
1823-1834.

Morgan MA, Wegner J, Aydelik E, Ganser A, and Reuter CWM (2002). Synergistic
cytotoxic effects in myeloid leukemia cells upon co-treatment with farnesyltransferase and
geranylgeranyl transferase-I inhibitors. Submitted to Leukemia.

Book Article

Reuter CWM, Morgan MA, and Bergmann L (2001). Effect of mutationally activated Ras
on the Ras to MAP kinase signaling pathway and growth inhibition of myeloid leukemia
cells by inhibitors of the MAP kinase cascade. In: Acute Leukemias VIII: Prognostic
factors and treatment strategies. Eds. Büchner T, Hiddemann W, Ritter J, Wörmann B,
Springer Verlag, Berlin, Heidelberg, New York, Tokyo.




I declare and certify herewith, that this work has been conducted by myself, without
employing unauthorized procedures or materials, and that it has not been submitted to any
other university or elsewhere in order to obtain an academic grade.

Michael Alexander Morgan
3
CONTENTS Page
1. INTRODUCTION........................................................................................... 7
1.1. The Ras family of GTP-binding proteins....................................................... 7
1.2. Post-translational modification of Ras.......................................................... 8
1.3. The Ras-to-MAP kinase signal transduction pathway.................................. 11
1.4. The Ras-to-Ral and the Ras-to-PI-3 kinase signaling pathways.................. 16
1.5. Role of Ras activation in hematological malignancies................................. 17
1.6. Inhibitors of the Ras-to-MAP kinase pathway.............................................. 21
1.7. Ras-signaling and effects of inhibitors of Ras-signaling in
myeloid leukemias........................................................................................... 30

2. MATERIALS AND METHODS.................................................................... 32
2.1. Materials.......................................................................................................... 32
2.1.1. Reagents and Solutions.............................................................................. 32
2.1.2. Cell lines...................................................................................................... 33
2.1.3. Antibodies.................................................................................................... 33
2.1.4. Inhibitors of Ras processing and signaling............................................... 33
2.1.5. Plasmid containing the c-Raf-1 domain that binds to activated Ras........ 34
2.2. Methods........................................................................................................... 34
2.2.1. Mammalian cell culture.............................................................................. 34
2.2.2. Trypan blue exclusion assay....................................................................... 34
2.2.3. Colony forming assays................................................................................ 34
2.2.4. Sequencing of Ras mutations..................................................................... 35
2.2.5. Western blot analysis.................................................................................. 38
2.2.6. MAP kinase assays...................................................................................... 39
42.2.7. Ras-GTP pulldown assay............................................................................ 40
2.2.8. Cell cycle analysis....................................................................................... 41
2.2.9. Immunocytochemical staining................................................................... 41
2.2.10. Detection of apoptosis................................................................................. 41
2.2.11. Proliferation assay of primary cells from leukemia patients................... 42
2.2.12. Analysis of combined drug effects. ............................................................ 43

3. RESULTS......................................................................................................... 44
3.1. Activation of the Ras-to-MAP kinase cascade............................................... 44
3.1.1. Ras mutations.............................................................................................. 44
3.1.2. Ras activation assays.................................................................................. 45
3.1.3. Activation of the MAPK cascade................................................................ 45
3.1.4. Activation of transcription factors............................................................. 49
3.1.5. Intracellular localization of PP-ERK-1/2 and PP-MEK-1/2.................... 49
3.1.6. MEK activation during cell cycle progression........................................... 49
3.2. Effects of Ras-to-MAPK signaling inhibitors in myeloid
leukemia cells.............................................................................................. 55

3.2.1. Effect of inhibitors of Ras-to-MAPK signaling on myeloid
leukemia cell growth....................................................................... 55

3.2.2. Inhibition of myeloid leukemia cell growth by Ras signaling
inhibitors is concentration dependent............................................ 55

3.2.3. Effect of inhibitors of Ras-to-MAPK signaling on cell cycle
progression...................................................................................... 60

3.2.4. Apoptosis induction by Ras signaling inhibitors....................................... 66

3.2.5. Effects of FTI/GGTI co-treatment on myeloid leukemia
cell growth....................................................................................... 69

3.2.6. Effects of FTI L-744,832 and GGTI-286 on Ras prenylation.................. 73

3.2.7. -286 on prenylation of
non-Ras proteins............................................................................ 73
5
3.2.8. Effects of FTI L-744,832 and GGTI-286 on Ras activation..................... 75
3.2.9. Effect of FTI/GGTI co-treatment on primary AML cells......................... 75


4. DISCUSSION................................................................................................... 82
4.1. Role of Ras in myeloid leukemias.................................................................. 82
4.2. Activation of Ras signaling in myeloid leukemias......................................... 83
4.3. Ras and the cell cycle...................................................................................... 84
4.4. Effects of Ras-signaling inhibitors in myeloid leukemia..............................84

5. REFERENCES................................................................................................ 92

6. ABBREVIATIONS...............................................................................