Characterizing the role of the Retinoblastoma protein LXCXE binding cleft in cellular senescence and tumor suppression

ii Co-Authorship Statement . iii Acknowledgments . iv Table of Contents . viii List of Figures . xiii List of Appendices . xv Chapter 1 . 1 1 Introduction . 1 1.1 Cancer, and the discovery of the first tumor suppressor gene RB1 . 1 1.1.1 The retinoblastoma 1 gene product (pRB) is a cell cycle regulator . 2 1.2 The pocket protein family . 2 1.3 The E2F family of transcription factors . 5 1.4 Cell cycle regulation by the pocket proteins . 7 1.4.1 Overlapping and unique roles of individual pocket proteins . 9 1.5 pRB’s unique role in tumor suppression . 11 1.6 A structure function approach to studying pRB function . 12 1.7 Gene targeted mice to study the contribution of the LXCXE binding cleft to pRB function . 13 1.8 Cellular senescence . 16 1.8. 1 Biomarkers of senescence . 16 1.8. 2 Types of senescence . 17 1.8. 3 Chromatin changes during senescence . 19 1.8. 4 The PML gene and PML nuclear bodies in senescence . 21 1.8. 5 Differences between human and mouse fibroblasts undergoing senescence . 24 ix 1.8. 6 Oncogene induced senescence in vivo . 26 1.8. 7 pRB’s role in senescence . 26 1.9 Objectives of the present study . 28 1.10 References . 29 Chapter 2 . 44 2 A G1 checkpoint mediated by the retinoblastoma protein that is dispensable in terminal differentiation but essential for senescence . 44 2.1 Abstract . 44 2.2 Introduction . 44 2.3 Results . 47 2.3.1 The Rb1 mutation causes defects in a senescent cell cycle arrest . 47 2.3.2 Permanent cell cycle exit during development is normal in Rb1 mice . 48 2.3.3 Defective senescence in Rb1 fibroblasts contributes to immortalization . 55 2.3.4 Incomplete senescence in Rb1 cells is characterized by defective transcriptional repression. 58 2.3.5 Rb1 cells fail to heterochromatinize E2F target gene promoters in senescence. 60 2.4 Discussion . 64 2.5 Materials and Methods . 68 2.5. 1 Mice . 68 2.5. 2 Cell culture . 68 2.5. 3 Histology and Fluorescence Microscopy . 69 2.5. 4 Quantitation of DNA, protein, and mRNA . 69 2.6 References . 70 Chapter 3 . 76 3 The Retinoblastoma protein and PML collaborate to organize heterochromatin and silence E2F responsive genes during senescence . 76 x 3.1 Abstract . 76 3.2 Introduction . 76 3.3 Results . 80 3.3.1 Defective enrichment of PML on E2F target gene promoters in Rb1 cells during senescence . 80 3.3.2 Early events during senescence induction occur normally in Rb1 MEFs . 82 3.3.3 Defective senescence arrest in Rb1 MEFs expressing PML-IV . 84 3.3.4 Induction of senescence signals the assembly of PML-pRB complexes that are essential for heterochromatin formation in senescence . 87 3.3.5 Defective chromatin compaction in Rb1 MEFs undergoing senescence . 91 3.4 Discussion . 93 3.5 Material and Methods . 96 3.5. 1 Cell culture . 96 3.5. 2 Immunofluorescence . 96 3.5. 3 mRNA expression analysis . 97 3.5. 4 GST pull downs and Immunoprecipitations . 97 3.5. 5 Chromatin Immunoprecipitations . 97 3.5. 6 Antibodies . 98 3.6 References . 98 Chapter 4 . 106 4 Mutation of the LXCXE binding cleft of pRB allows escape from oncogenic ras induced senescence and transformation in vitro but does not promote tumorigenesis in vivo . 106 4.1 Abstract . 106 4.2 Introduction . 107 4.3 Results . 110