Effects of Ras Oncoprotein

Effects of Ras Oncoprotein - Effects of Ras Oncoprotein and...

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Effects of Ras Oncoprotein and Growth Factors on the Cell Cycle Regulators Retinoblastoma Protein Rb and p21 Cip1/Waf1 Laura Kivinen Department of Virology Haartman Institute University of Helsinki Finland Helsinki 1999
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Effects of Ras Oncoprotein and Growth Factors on the Cell Cycle Regulators Retinoblastoma Protein Rb and p21 Cip1/Waf1 Laura Kivinen Department of Virology Haartman Institute University of Helsinki Finland Academic Dissertation To be presented, with the permission of the Medical Faculty of the University of Helsinki, for public criticism in the Small Lecture Hall of Haartman Institute, Haartmaninkatu 3, Helsinki, on October 1st, 1999, at 12 noon Helsinki 1999
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Supervised by Docent Marikki Laiho Department of Virology Haartman Institute University of Helsinki Finland Reviewed by Docent Mathias Bergman Department of Biosciences University of Helsinki Finland and Docent Erkki Hölttä Department of Pathology University of Helsinki Finland Opponent Docent Lea Sistonen Turku Centre of Biotechnology Finland
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ISBN 951-45-8695-6 (PDF version) Helsingin yliopiston verkkojulkaisut Helsinki 1999
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4 CONTENTS ORIGINAL PUBLICATIONS__________________________________________6 ABBREVIATIONS ___________________________________________________7 SUMMARY _________________________________________________________8 INTRODUCTION___________________________________________________10 REVIEW OF THE LITERATURE_____________________________________12 Cell cycle and G 1 restriction point control ___________________________________________12 Ras-proteins, important transmitters of extracellular signals ___________________________14 The RAS family _________________________________________________________________14 Activation of H-Ras______________________________________________________________14 Inactivation of H-Ras ____________________________________________________________15 Cellular transformation by Ras ____________________________________________________16 Ras and cell cycle regulation ______________________________________________________18 Ras in differentiation, growth arrest and senescence ____________________________________19 The mitogen-activated protein kinase pathways________________________________________21 Ras and the mitogen-activated protein kinase pathways _________________________________23 Other pathways activated by Ras ___________________________________________________25 Retinoblastoma tumor suppressor__________________________________________________27 Functions of the retinoblastoma protein (Rb)__________________________________________27 Regulation of Rb-activity by phosphorylation__________________________________________27 Binding of Rb to its targets ________________________________________________________28 Rb and cell cycle control _________________________________________________________29 The Rb protein family ____________________________________________________________30 p21 cyclin kinase inhibitor ________________________________________________________32 p21 inhibits the activity of Cdks ____________________________________________________32 p21 and cell cycle regulation ______________________________________________________32 p21 in differentiation, senescence and transformation___________________________________33 p21 induction by p53_____________________________________________________________34 p53-independent regulation of p21__________________________________________________35 Transcriptional and posttranscriptional regulation of p21 _______________________________35 OUTLINE OF THE STUDY __________________________________________38 MATERIALS AND METHODS _______________________________________40 Cell culture, transfections and soft agar assays _______________________________________40 Chemicals and growth factors _____________________________________________________41 Protein analyses_________________________________________________________________41 Immunofluorescence_____________________________________________________________41 Immunoblotting_________________________________________________________________41 Immunoprecipitation_____________________________________________________________42 mRNA analysis by Northern blotting _______________________________________________42 DNA analysis by Southern blotting _________________________________________________42 cDNA probes ___________________________________________________________________43 p21-promoter constructs and luciferase assay ________________________________________43
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5 Electrophoretic mobility shift assays (EMSA) ________________________________________43 Cell cycle analyses_______________________________________________________________44 FACS-analysis _________________________________________________________________44 5-bromo-2'-deoxy-uridine incorporation _____________________________________________44 RESULTS AND DISCUSSION ________________________________________45 Human wild-type Rb prevents c-Ha-Ras transformation in NIH 3T3 cells (I, II)____________45 Mutual regulation of exogenous Rb and c-Ha-Ras expression in NIH 3T3 cells (I, II) __________45 Pathways regulating Rb by c-Ha-Ras (I, II) ___________________________________________47 Mechanisms for inhibition of c-Ha-Ras transformation by Rb (I, II) ________________________48 Relevance of mutual regulation of Rb and Ras (I, II) ____________________________________48 c-Ha-Ras-regulation of other target genes in Rb-expressing NIH 3T3 cells (II) _______________49
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Effects of Ras Oncoprotein - Effects of Ras Oncoprotein and...

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